Compositions and methods for modulating connexin hemichannels

ABSTRACT

Disclosed are compositions and methods for modulating hemichannel function in a cell, tissue or organ. The invention also relates to useful screens for detecting such compounds, particularly those capable of modulating connexin phosphorylation. Further provided are therapeutic methods for preventing or treating conditions impacted by undesired hemichannel function in a mammal such as heart arrhythmia.

CROSS-REFERENCE TO RELATED APPLICATION

The present application is a divisional of U.S. patent application Ser.No. 10/353,549, filed on Jan. 29, 2003, which is a continuation-in-partof U.S. Provisional Application Ser. No. 60/352,717, filed on Jan. 29,2002, all of which are hereby incorporated by reference.

FIELD OF THE INVENTION

The present invention generally relates to compositions and methods formodulating connexin hemichannels. The invention also relates to usefulscreens for detecting such compounds.

BACKGROUND

There is recognition that gap junctions are important plasma membranestructures that help cells communicate with their environment. Forexample, most gap junctions are thought to assist passage of smallmolecules and ions between interconnected cells. Such movement isbelieved to exert profound effects on many aspects of cell physiology.Plasma membranes of adjacent cells are believed to include hemichannels,“connexons”, formed by multimeric proteins called “connexins” that helpform the gap junctions. In addition, hemichannels play an independentrole in the exchange of small molecular weight compounds between thecell cytoplasm and the periplasmic or extracellular space. See generallyBennett, M. et al. (1991) Neuron 6: 305; Kumar, N. and Gilula, N. B.(1996) Cell 84: 381; and Quist, A. P et al (2000) J. Cell Biol. 148:1063 and references cited therein.

In particular, there have been reports that many gap junctions arespecialized regions of the cell membrane with clusters of densely packedchannels. Such gap junction channels are thought to directly connect thecytoplasmic compartment of two neighbouring cells.

There is recognition that gap junction channels are composed of twohemichannels (connexons) provided by each of two neighbouring cells.Each connexon (hemichannel) has been disclosed as consisting of sixproteins called connexins. Each connexin is thought to share fourtransmembrane domains, two extracellular loops, and a cytoplasmic loop.The conduction of the electrical impulse is thought to take placethrough the gap junctions, thereby facilitating normal heart conductionand rhythm. See generally P. A. Guerrero, R. B. et al. J Clin Invest1997, 99 1991; D. L. Lerner, K. A. et al., Circulation 1999, 99 1508; S.Kirchhoff, E. et al. Curr Biol 1998, 8 295.

Distribution of most heart connexins is thought to vary significantlythroughout the organ. It has been disclosed that the Cx43 isoform is amajor ventricular type while Cx40 is the most abundant isoform in theatrias and conductive system.

There are reports of strong links between connexin abnormalities andheart disease. See A. C. de Carvalho, et al., J CardiovascElectrophysiol 1994, 5 686; R. R. Kaprielian, et al., Circulation 1998,97 651; N. S. Peters, et al., Circulation 1993, 88 864; and J. E.Saffitz, R. B. et al., Cardiovasc Res 1999, 42 309.

There is understanding that abnormal expression, distribution andregulation of gap junctions are involved in arrhythmias. Theantiarrhythmic peptides disclosed by Larsen, B. et al. in PCT/DK01/00127(WO01/62775) have been reported to increase gap junction intercellularcommunication (GJIC) in vertebrate tissue.

Particular mammalian gap junction proteins encoded by the connexin (Cx)gene family have been reported. See Bruzzone, R. et al. (1996) Eur. J.Biochem. 238: 1. The Cx family includes Cx26, 30, 31, 32, 37, 40, 43,45, 46 and 50. Gap junction channels have also been found ininvertebrates, where the channel forming proteins are called “innexins”

There is understanding that most connexins may be phosphorylated exceptfor the Cx26 protein. The Cx43 protein is widely expressed in tissues.There are reports that phosphorylation of the Cx43 protein effects gapjunction intracellular communication (GJIC). For example, there isacknowledgement that Cx43 turn over, trafficking, phosphorylation andgating are impacted by phosphorylation. See Darrow, B. J., et al.(1995). Circ Res 76: 381.

For example, Saffitz and co-workers have shown using conductancemeasurements that during ischemia there is an increase in connexinserine dephosphorylation within 15 minutes. See FIG. 1 (showing amammalian Cx43 transmembrane protein with several identifiedphosphorylation sites).

The phosphorylation and solubility of the connexins has attractedresearch interest. In particular, Cx43 was found to be phosphorylated inthe myoepithelial cells of rat mammary glands. See Wang, Y., et al.(1995). J Biol Chem 270, 26581; and Yamanaka, I., et al. (1997). Eur JCell Biol 72: 166.

As mentioned, gap junction channels are thought to be specialized poresthat connect the cytoplasm of neighboring cells. Hemichannelscommunicate with the extracellular environment. There have been reportsthat metabolic inhibition of heart cells can activate an influx pathwaythat may be structured by connexin hemichannels. Metabolic inhibition isthought to open the hemichannel and enhance loss of potassium, andinduce influx of protons, sodium and calcium, thereby damaging hearttissue. See Kondo et al J. Mol. Cell. Cardiol. 32:1859-72, 2000; Li etal J. Mol. Cell. Cardiol. 33: 2145-55, 2001).

Electrical uncoupling at gap junctions during acute myocardial ischemiais believed to contribute to conduction abnormalities and reentrantarrhythmias. Increased levels of intracellular Ca²⁺ and H⁺ andaccumulation of amphipathic lipid metabolites during ischemia promoteuncoupling. Other mechanisms may play a role. For instance, it has beenreported that uncoupling induced by acute ischemia is associated withchanges in phosphorylation of connexin43 (Cx43). Results have beenreported that are consistent with rapid, reversible Cx43dephosphorylation playing a role in myocardial uncoupling andarrhythmogenesis during acute ischemia. See Beardslee M A et al., CircRes. 2000; 87:656 and references cited therein.

The structure and function of hemichannels have attracted interest.

For example, atomic force microscopy (AFM), fluorescent dye uptakeassay, and laser confocal immunofluorescence imaging, have suggestedthat hemichannels are involved in extracellular calcium-dependentmodulation of cell volume. As reported, cell volume changes weredependent on whether or not connexin43 was expressed. Changes werereported to be preventable by gap-junctional blockers (e.g., oleamideand beta-glycyrrhetinic acid) or were reversed by returningextracellular calcium to normal. It was suggested that nongap-junctionalhemichannels regulate cell volume in response to the change inextracellular physiological calcium in an otherwise isosmotic situation.See Quist, A. P. et al, supra.

It has been proposed that open hemichannels, especially during ischemiaor metabolic stress, may lead to cellular uptake of Ca²⁺, protons andaccumulation of amphipathic lipid metabolites in cells causing cellularswelling, cell damage or apoptosis. See Beardslee et al., supra andQuist et al. supra.

There have been reports Cx43 is phosphorylated a positions Tyr247(Y247), Tyr265 (Y265) and perhaps other positions by the activated Srcprotein in vitro. Significantly, gap junction intercellularcommunication (GJIC) was reported to be resistant to disruption byphosphorylation mediated by v-Src. It was acknowledged thatphosphorylation on Y247 and Y265 of Cx43 is important. See Lin R, et al.(2001) J Cell Biol 154(4):815. See also FIG. 1.

See also Larsen, B. D et al. in a PCT application entitled New MedicalUses of Intracellular Communication Facilitating compounds as filed on22 Feb. 2002 as PCT/US02/05773 (WO 02/077017) in which a variety of GJICmodulating compounds have been disclosed.

It would be desirable to have compounds that modulate hemichannelfunction. Preferred compounds would assist opening or closing of thehemichannel. It would be especially desirable to have molecular screensto identify such compounds.

SUMMARY OF THE INVENTION

The invention generally features compounds and methods of using same tomodulate hemichannel function. Particular compounds modulate hemichannelphosphorylation. Additional compounds of the invention modulatehemichannel function and in some instances also impact gap junctioncommunication (GJIC) eg., by opening or closing gap junction channels.Useful screens for detecting and characterizing such compounds are alsoprovided. The invention has many useful applications including providingtherapies to treat or prevent various conditions modulated by unsuitablehemichannel function.

There is an urgent need to identify compounds that modulate (increase ordecrease) hemichannel function. By “hemichannel function” is meant theopening or closing of connexin hemichannels to enhance or decreasepassage of molecules or ions through the hemichannel. By the phrase “gapjunction function” is meant the opening or closing of gap junctions toincrease or reduce passage of molecules or ions through the gapjunction. Preferred invention compounds modulate hemichannelphosphorylation and, typically also help open or close the hemichannel.Hemichannel function and gap junction function can be readily detectedand optionally quantified by one or more of the standard assaysdisclosed herein.

More specific hemichannel modulating compounds modulate (increase ordecrease) phosphorylation of a recognized connexin. Preferred sites ofphosphorylation or dephosphorylation include one or more of a tyrosine,serine or threonine residue on the connexin. As will be discussed below,it has been found that modulation of phosphorylation on one or more ofthese residues impacts hemichannel function, particularly by opening andclosing the hemichannel. Thus, and as described below, it is an objectof the invention to provide screens adapted to monitor thephosphorylation state of the connexins as a means of detecting andoptionally characterizing compounds with capacity to modulatehemichannel function.

For instance, certain compounds according to the invention are capableof phosphorylating at least one tyrosine residue of the connexin. Inthis embodiment, phosphorylation of the connexin will help close thehemichannel. Other suitable hemichannel modulating compounds decreasephosphorylation (dephosphorylate) at least one serine residue of theconnexin. In this example, dephosphorylation of the serine will helpopen the hemichannel. Still other compounds within the scope of theinvention will enhance phosphorylation of at least one threonine residueof the connexin, typically to assist in the closure of the hemichannel.Additionally suitable invention compounds facilitate at least one of: anincrease or decrease in serine phosphorylation, an increase or decreasein tyrosine phosphorylation, and an increase or decrease in threoninephosphorylation of the connexin. Preferably, one or more of the aminoacid modifications will assist in a detectable opening or closing of thehemichannel.

Accordingly, and in one aspect, the invention provides a method tomodulate hemichannel function preferably by closing the hemichannel. Inone embodiment, the method involves closing the hemichannel in a cell,tissue or organ preferably exposed to stress including contacting thestressed cell, tissue or organ with a therapeutically effective amountof at least one of the compounds represented below as Formula I or II.Preferred contact according to this method embodiment is sufficient toclose the hemichannel before, during or after exposure to the stressrelative to a suitable control. Examples of such stress include one ormore of metabolic inhibition, oxygen deprivation, lowering pH, orincreasing extracellular potassium ion as described below.

In a more specific embodiment, the method further includes monitoringphosphorylation of a recognized connexin, preferably connexin 43 (Cx43). Typically, the method will detect and report any increase ordecrease in Cx43 phosphorylation on at least one of a tyrosine, serine,and threonine reside thereon, preferably an increase in phosphorylationin at least one of tyrosine and threonine. In this embodiment, themethod also includes closing the hemichannel and, optionally, opening orclosing gap junction channels relative to a suitable control.

The invention provides other methods to modulate hemichannel function.In one embodiment, the method involves monitoring phosphorylation of arecognized connexin, preferably Cx43, to detect any increase or decreasein Cx43 phosphorylation on at least one of a tyrosine, serine andthreonine residue thereon, preferably a decrease in phosphorylation ofone or more of those residues such as serine relative to a control. Inthis invention example, the method involves opening the hemichannel in acell, tissue or organ exposed to stress including contacting thestressed cell, tissue or organ with a therapeutically effective amountof at least one of the compounds represented below as Formula I or II.Preferred contact is sufficient to open the hemichannel and, optionally,open or close gap junction channels relative to a suitable control.

There is a need for screens to detect and characterize such hemichannelmodulating compounds more specifically. Having such screens would be animportant first step toward identifying and characterizing a range ofnew hemichannel modulating compounds e.g., by in vitro testing of acandidate compound relative to a control to detect capacity to helpclose hemichannels, for instance, by at least 5% more than a controlcompound. compounds identified by such a screen, including the compoundsrepresented below as Formula I or II, can be used to in therapies thatpromote cell, tissue and organ homeostasis, for instance, by preventing,reducing or protecting against loss of cellular components to theextracellular environment.

Accordingly, the present invention also provides specific in vitromethods for screening candidate compounds that have capacity to modulatehemichannel function. Typically, the method involves contacting suitablecells, tissue or an organ with at least one compound represented belowas Formula I or II. Preferably, the contact is under conditionsconducive to modulating phosphorylation of a recognized connexin,preferably connexin 43 (Cx43) and detecting a change in Cx43phosphorylation relative to a suitable control. Also preferably, thechange in phosphorylation is taken to be indicative of a hemichannelmodulating compound. Optionally, such compounds detected by thescreening method may open or close gap junction channels according toassays disclosed herein.

In each of the foregoing methods, preferred phosphorylation changesaccording to the invention occur almost entirely at or near theintracellular C-terminus of the connexin. More preferred sites ofphosphorylation and dephosphorylation of Cx43 are shown in FIG. 1. Bythe phrase “C-terminus of connexin” is meant the region spanning aboutamino acid residues 240 to 281 as shown in FIG. 1.

A particular in vitro screening assay of the invention for detectingconnexin phosphorylation involves one or more steps designed to monitorhemichannel function, gap junction function (or both). Such an assaygenerally includes at least one and preferably all of the followingsteps:

-   -   1) culturing a population of cells, a tissue or an organ such as        those derived from the heart or muscle,    -   2) stressing the cells, tissue or the organ preferably by oxygen        deprivation or metabolic inhibition,    -   3) adding a known or candidate hemichannel modulating compound        such as those represented by Formula I or II below,    -   4) detecting a change in connexin phosphorylation (preferably        Cx 43) relative to a suitable control; and    -   5) optionally measuring the change as being indicative of a        compound that modulates hemichannel function and optionally gap        junction function.

That assay can effectively measure capacity of the hemichannelmodulating compound to increase or decrease phosphorylation of at leastone of a serine, tyrosine and threonine residue of the preferred Cx43protein. Reference herein to a “standard in vitro connexinphosphorylation assay” or related phrase refers to the above protocol ofsteps 1) through 5). The assay can be conducted with nearly anypopulation of primary, secondary, or immortalized cells such as thosederived from heart or muscle.

The foregoing standard in vitro assay is generally flexible. Forinstance, steps 1)-5) can be performed in nearly any order providedintended screening results are achieved. Thus in one embodiment of theassay, the candidate compound is added at step 1), step 2) (or bothsteps) instead of after step 2) exclusively.

The present invention provides other methods for screening candidatecompounds for capacity to modulate hemichannel function. In oneembodiment, the method includes contacting suitable cells, tissue or anorgan with at least one compound selected from the group representedbelow as Formula I or II. Typically, any uptake of a detectable reporterby the cells, tissues or organ is monitored in the presence of thecompound and relative to a suitable control. Preferred detectablereporters have a molecular size that is conducive to passage through anopen hemichannel. Thus, when the hemichannel is open in the assay, thedetectable reporter enters the cell, tissue or organ. However when thehemichannel is closed, the detectable reporter is prevented from passingthrough the hemichannel. The contact is preferably under conditionsconducive to detecting any change in the uptake of the detectablereporter with reference to a suitable control.

A hemichannel is “closed” in accord with the invention if at least oneof the tyrosine residues in the C-terminal region of connexin,preferably Cx43 is phosphorylated, preferably at least the tyrosine atposition 247 or position 265, more preferably both of same, as detectedfor instance in the standard in vitro connexin phosphorylation assay. By“closed” is also meant at least one of the threonine residues in theC-terminal region of the connexin is phosphorylated, which residues maybe phosphorylated alone or in addition to tyrosine phosphorylation. Ahemichannel is “open” if at least one of the serine residues in theC-terminal region of the connexin is dephosphorylated. Additionallypreferred tyrosine, threonine and serine residues are shown in FIG. 1 askinase sites.

A more specific in vitro screening assay for detecting passage of thedetectable reporter through the hemichannel involves one or more of thefollowing steps:

-   -   1) culturing a population of cells, a tissue or an organ such as        those derived from the heart or muscle,    -   2) stressing the cells, tissue or the organ preferably by oxygen        deprivation or metabolic inhibition such as by adding a glucose        derivative,    -   3) adding a known or candidate hemichannel modulating compound        such as those represented below by Formula I or II,    -   4) adding a detectable reporter such as a fluorescent,        chemiluminescent, or phosphorescent compound such as fluorescent        dye such as calcein and related compounds,    -   5) detecting a change in uptake of the detectable reporter into        the cells, tissue or organ relative to a suitable control; and    -   6) optionally measuring the change as being indicative of a        compound that modulates hemichannel function.

That assay can effectively measure capacity of the candidate compound toopen or close hemichannels in the cells, tissue or organ which change isreadily detectable microscopically by visualizing the detectablereporter. Reference herein to a “standard in vitro uptake assay” orrelated phrase refers to the above protocol of steps 1) through 6). Theassay can be conducted with nearly any population of primary, secondary,or immortalized cells such as those derived from heart or muscle. Otheracceptable reports include suitable radioactive compounds also having asize permitting passage through the hemichannel. Particular compoundsinclude those labeled with one or more of the following radionuclides:³H, ³⁵S, and ¹⁴C.

Importantly, the standard in vitro uptake assay described generallyabove is not bound to any particular order of steps so long as intendedassay results are achieved. Thus in one embodiment, at least one of thecandidate compound and detectable reporter of steps 3) and 4),respectively, are added individually or together before step 2) in themethod. In this example of the assay, the cells, tissue or organ isstressed in the presence of the compound and the detectable reporter.Alternatively, the detectable reporter can be “loaded” into the cells atstep 1) to detect compounds with capacity to open hemichannels.

The invention provides further methods for screening one or morecandidate compounds for capacity to modulate hemichannel function. Inone embodiment, the method includes contacting cells, tissue or an organwith at least one compound selected from those represented by Formula Ior II below. Typically, the cells, tissue or organ is loaded with adetectable reporter to measure volume. Preferred detectable reportersfor use in the assay have a molecular size that is suited for passingthrough an open hemichannel. The contact is preferably under conditionsconducive to detecting any change in the volume of the cells, tissue ororgan as registered by the detectable reporter and by referring to asuitable control. Preferably, the cells, tissue or organ is stressed andthe candidate compound closes the hemichannel so that cell volume ismaintained or decrease more slowly when compared to a suitable control.

One particular in vitro screening assay for detecting cell volumechanges involves one or more of the following steps:

-   -   1) culturing a population of cells, a tissue or an organ such as        those derived from the heart or muscle,    -   2) loading the cells, tissue or organ with a detectable reporter        such as a fluorescent, chemiluminescent, or phosphorescent        compound such as dye, such as calcein or a related compound,    -   3) estimating the volume of the cells, tissue or organ by        detecting and quantifying signal from the detectable reporter,    -   4) adding a known or candidate hemichannel modulating compound        such as those represented by Formula I or II below,    -   5) stressing the cells, tissue or the organ preferably by oxygen        deprivation or metabolic inhibition such as by adding a glucose        derivative,    -   6) detecting a change in cell volume relative to a suitable        control; and    -   7) optionally measuring the change as being indicative of a        compound that modulates hemichannel function and optionally gap        junction function.

The assay can effectively measure capacity of the candidate hemichannelmodulating compound to open or close hemichannels in the cells, tissueor organ by observing cell volume changes microscopically. Referenceherein to a “standard in vitro cell volume assay” or related phraserefers to the above protocol of steps 1) through 7). The assay can beconducted with nearly any population of primary or secondary cellsderived from heart or muscle such as cardiomyocytes and related cells ortissue.

The standard in vitro cell volume assay is not bound to any particularorder of steps so long as intended assay results are achieved. Thus inone embodiment, the candidate compound of step 4) is added afterstressing the cells in step 3) to monitor ability of the compound toclose hemichannels in already stressed cells as exemplified by a slowerrate of cell volume decrease. Thus in one embodiment of the assay, achange in the rate of cell volume decrease or increase is monitored overa pre-determined time frame. However in another embodiment, the cellvolume change can be monitored at a fixed time point e.g., at a timebetween about 1 to 120 minutes after stressing the cells, tissue ororgan.

As discussed, the in vitro assays of the invention are flexible and canbe adapted to suit an intended screening use. For instance, a particularcandidate hemichannel modulating compound, such as those represented byFormula I or II below, can be employed as the sole active agent or incombination with other agents including other compounds to be tested. Inmost, but not all instances, the in vitro assays are performed withreference to a suitable control assay usually including the same orclosely related test conditions as in the steps above, but withoutadding the compound to be tested to the culture medium. In such cases, acandidate hemichannel modulating compound can be identified byexhibiting at least 2% greater activity in the assay relative to thecontrol, more preferably at least about 5% greater activity relative tothe control assay, and still more preferably at least about 10% orgreater activity, eg., about 20% to about 40% relative to the controlassay.

As discussed, particular hemichannel modulating compounds will alsoimpact gap junction channels. For instance, certain compounds may helpclose the hemichannels and assist in the opening of gap junctionchannels. However, other compounds may help open the hemichannels whileassisting in the closure of gap junction channels. Still other compoundsmay open both hemichannels and gap junctions while others may close gapjunctions and hemichannels.

Accordingly, the invention also provides a method of increasing gapjunction intracellular communication (GJIC) in a cell, tissue or organ.In one embodiment, the method involves administering a therapeuticallyeffective amount of at least one compound selected those represented byFormula I or II as described below. Preferably, the contact issufficient to increase the GJIC in the cell, tissue or organ relative toa suitable control.

Also provided are combinations of in vitro assays in compounds areselected for capacity to modulate hemichannels and gap junctions. In oneembodiment, at least one of the standard in vitro connexinphosphorylation assay, the standard in vitro uptake assay, and thestandard in vitro cell volume assay, is combined with one or more of theGJIC assays disclosed by Larsen, B. et al. in a PCT application entitledNovel Antiarrhythmic Peptides as filed on 22 Feb. 2001 as PCT/DK01/00127(WO 01/62775) or as disclosed by Larsen, B. et al. in another PCTapplication entitled New Medical Uses of Intracellular CommunicationFacilitating compounds as filed on 22 Feb. 2002 as PCT/US02/05773 (WO02/077017). Examples of such suitable GJIC assays include thosemeasuring cell conductance by patch clamp, calcium wave measurements anddye transfer assays. The disclosures of the PCT/DK01/00127 (WO 01/62775)and PCT/US02/05773 (WO 02/077017) applications are hereby incorporatedby reference.

By way of illustration and not limitation, the standard in vitroconnexin phosphorylation assay described above is employed to select oneor more hemichannel modulating compounds. One or more the compounds cansubsequently be further screened in the cardiomyocyte patch clamp assaydescribed in the PCT/US02/05773 (WO 02/077017) application. compoundsproviding suitable activity in both assays will have capacity tomodulate hemichannels and gap junctions.

The invention further provides in vivo testing of the candidatehemichannel modulating compounds to help detect and optionally quantifytherapeutic capacity to modulate a heart arrhythmia. As discussed, it isbelieved that most heart arrhythmias can be prevented, alleviated ortreated by use of one or a combination of the compounds of thisinvention. A preferred in vivo testing model, referred to herein as a“standard in vitro mouse arrhythmia assay” or related phrase, has beendisclosed in PCT/DK01/00127 (WO 01/62775) as well as in PCT/US02/05773(WO 02/077017). Certain compounds according to the invention willdesirably prolong the time until onset of induced atrial ventricular(AV) block by at least about 20% (score of at least 2) in the assay.Other compounds will exhibit a prolongation of at least about 60% or thetime until onset of the induced AV block (score of at least 3) in theassay. In broad terms, the assay involves administering one or morecompounds to a suitable mouse, injecting calcium chloride to inducearrhythmia, and detecting the time of onset of the arrhythmia(preferably 2^(nd) degree AV-block) compared to a suitable control.

Significantly, use of multiple detection formats (ie., a combination ofat least one of the standard in vitro assays and the in vivo arrhythmiaassay as disclosed herein) can efficiently perform multiple analyses,thereby enhancing the accuracy and probability of identifying ahemichannel modulating compound (as represented by Formulae I and II,for instance) with good therapeutic capacity. This feature of theinvention is especially useful when large numbers of compounds are to betested. For example, some or nearly all of the compounds according toFormula I or II can be tested. Alternatively, or in addition, suitablecompounds could be made by standard synthetic methods includingcombinatorial-type chemical manipulations and then tested in accord withthe invention.

In embodiments in which multiple detection formats are practiced, it isimportant to note that significant in vitro and in vivo activity asdetermined by the assays described herein is not a required feature of ahemichannel modulating compound. That is, certain compounds describedherein will exhibit good activity in at least one of the standard invitro assays described herein but will not exhibit significant activityin the standard in vivo arrhythmia assay. Alternatively, certain othercompounds will exhibit significant activity in the in vivo arrhythmiaassay but will not show good activity in one or more of the standard invitro assays. However, a preferred hemichannel modulating compound willexhibit good activity in at least one of the in vitro and in vivo assaysdescribed in this application.

Compounds showing good activity in the standard in vivo arrhythmia assaywill sometimes be called “antiarrhythmic compounds” or like phrase todenote capacity to prolong time to arrhythmia in the assay.

As will be discussed in more detail below, compounds of the inventioncan be used to prevent or treat a wide spectrum of medical conditionsthat are associated or suspected of being related to undesired orabnormal passage of molecules and/or ions through cell membranes. Forinstance, nearly all the medical indications disclosed in PCT/DK01/00127(WO 01/62775) and PCT/US02/05773 (WO 02/077017) can be addressed in thisway. Some of said medical indications are disclosed herein. The medicalindications may be prevented, alleviated or treated by using one or acombination of compounds disclosed in the present application andparticularly those showing good activity in at least one of the in vitroand in vivo assays provided herein.

Additional compounds suitable for testing and use with the presentinvention have been disclosed by Larsen, B. D et al. in PCT/US02/05773(WO 02/077017).

In another aspect, the invention provides a method of preventing ortreating tissue or organ stress in a mammal. In one embodiment, themethod includes administering a therapeutically effective amount of atleast one compound selected from the group of compounds represented byFormulae I and II above. Preferably, the contact is sufficient toprevent or treat the stress in tissue or organ.

The invention also provides for a method of treatment of burnscomprising administering to a patient in need of such treatment atherapeutically effective amount of a compound that blocks connexinhemichannel opening.

Also provided is a method of treatment of thromboses. In one embodiment,the method includes administering to a patient in need of such treatmenta therapeutically effective amount of a compound that blocks a connexinhemichannel from opening.

In another aspect, the invention also features a method of treatment ofrespiratory and metabolic acidosis. In one embodiment, the methodincludes administering to a patient in need of such treatment atherapeutically effective amount of a compound that blocks a connexinhemichannel from opening.

The invention also provides for a method of treatment of focalarrhythmia. In one example of the method, it includes administering to apatient in need of such treatment a therapeutically effective amount ofa compound that blocks a connexin hemichannel from opening.

Further provided by the invention is a method of treating and preventingcell and tissue damage resulting from elevated levels of blood glucose.In one embodiment, the method includes administering to a patient inneed of such treatment a therapeutically effective amount of a compoundthat blocks a connexin hemichannel from opening.

The invention also features a method of treatment of chronic atrialfibrillation. In one embodiment, the method includes administering to apatient in need of such treatment a therapeutically effective amount ofa compound that blocks a connexin hemichannel from opening.

Also provided is a method of treatment of epilepsia. Typically, themethod includes administering to a patient in need of such treatment atherapeutically effective amount of a compound that promotes a connexinhemichannel to open.

Further provided is a method of cytoprotecting tissue or an organ of amammal in need of such treatment, the method comprising administering atherapuetically effective amount of at least one compound selected fromthe group consisting of the compounds represented by Formula I or II.

The invention also provides for a method of preventing or treatingreperfusion injury in a mammal, the method comprising administering atherapuetically effective amount of at least one compound selected fromthe group consisting of the compounds represented by Formula I or II.

In each of the foregoing therapeutic methods, a compound according tothe invention that features good anti-arrhythmic activity as determinedby the standard in vitro mouse arrhythmia assay (ie., score of at least2). Such compounds will sometimes be referred to herein as“antiarrhythmic” compounds or a related phrase.

Further uses and advantages of the present invention will be apparentfrom the following discussion and examples. Other aspects of theinvention are also discussed below

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 (SEQ ID NO: 1) is a drawing showing phosphorylation sites on aconnexin (Cx43). As can be seen, the cytosolic domain of the Cx43transmembrane protein has several potential serine and tyrosinephosphorylation sites.

FIG. 2 is a graph showing metabolic stress induced by removal ofglucose. As can be seen from the graph, administration of compound 1reduces the stress as evidenced by a decrease in relative conductiondelay.

FIGS. 3A-B are representations of immunoblots showing detection oftyrosine phosphorylation in connexin 43 (Cx43). FIG. 3A shows resultsfrom compound 1 administration. FIG. 3B shows results fromadministration of compound 1 and compound 2.

FIGS. 4A-B are representations of immunoblots showing detection oftyrosine phosphorylation (8A) and serine phosphorylation (8B) of Cx43.

FIG. 5 is a graph showing effect of 10 nM compound 1 on ischemia-induceduptake of calcein in cultured cardiomyocytes.

FIG. 6A is a drawing showing a preferred ELISA assay format. FIG. 6B isa graph showing ELISA results of phosphorylated Cx43 at Tyr-P in HeLacells. FIG. 6C is a graph illustrating ELISA results of phosphorylatedCx43 at Tyr-P in CHO cells.

FIG. 7A-C are photomicrographs showing dye uptake in culturedcardiomyocytes. FIG. 7A: Cardiomyocytes under light microscopy; FIG. 7B:Fluorescence under control conditions (same as cells in FIG. 7A); FIG.7C Fluorescence after 30 minutes of metabolic inhibition.

FIG. 8 is a graph showing that compound 1 reduces stress-induced calceinuptake in a dose-dependent manner.

FIG. 9A-B are graphs showing effect of compound 1 on stress-induced cellswelling. FIG. 9A Relative volume during metabolic inhibition in thepresence or absence of compound 1 (0.1 nM). FIG. 9B shows control data.

FIG. 10 is a graph showing effect of compound 1 (0.1 nM) onstress-induced cell swelling.

FIG. 11 is a graph showing a reduction in heart weight to body weightratio after administration of Compound 1 to rats subjected to myocardialinfarction.

FIG. 12 is a graph showing a reduction in infarct size in rats followingadministration of Compound 1.

FIG. 13 is a graph showing improved cardiac function afteradministration of Compound 1 to rats subjected to myocardial infarction.

DETAILED DESCRIPTION OF THE INVENTION

As discussed, the invention provides compounds and methods of using sameto modulate hemichannel function. More particular compounds modulatehemichannel phosphorylation to assist opening or closing of hemichannel.Useful screens for detecting and characterizing such compounds are alsoprovided which screens include in vitro assays, an in vivo assay, or acombination thereof. Further provided are therapeutic methods useful forthe treatment or prevention of conditions impacted by unsuitablehemichannel function.

It is an object of the invention to demonstrate, for the first time,that cell, tissue and organ stress may be adversely impacted by theclosing and opening of hemichannels. Without wishing to be bound totheory, it is believed that metabolic stress, such as oxygen deprivationduring ischemia, glucose deprivation, uncoupling of the oxidativephosphorylation caused by HCN, or uncoupling of the citric acid cycle,may all contribute to uncoupling of the intercellular gap junctionalcommunication (GJIC). It is further believed that this uncoupling iscorrelated with modulation of connexin phosphorylation, moreparticularly dephosphorylation of connexin-tyrosine residues and/orconnexin-serine residues and/or threonine residues in the gap junctionchannel. By way of illustration, it is believed that during atrialfibrillation, the atrial cells have increased metabolic demand due tothe high frequency pacing. This is thought to lead to lactate acidosis,opening of hemichannels and uncoupling of gap junctions.

It is also an object of this invention to provide methods of modulatinghemichannel function and, optionally, gap junction intercellularcommunication (GJIC) which methods include contacting cells, tissue ororgans with a therapeutically effective amount of one or a combinationof compounds that have such activity. Preferred compounds have beendisclosed in the PCT/DK01/00127 (WO 01/62775) and PCT/US02/05773 (WO02/077017) by B. Larsen et al.

More preferred compounds suitable for use with the present inventioninclude those represented by the following Formula I:

whereinR1 represents H or acetyl (Ac)R2 represents a sidechain of one of the amino acids G, Y, D-Y, F andD-F,R3 represents any amino acid sidechain R4 represents a sidechain of oneof the amino acids G, Y, D-Y, F and D-F,R5 represents OH or NH2and a, S, T, P and Q are integers are integers and independently=0 or 1;and salts thereof.More specific compounds include those having the following Formula II:R1-X1-X2-X3-R2  IIwherein,

X1 is 0, Ala, Gly, β-Ala, Tyr, D-Tyr, Asp,

X2 is 0; Ala-Gly-T4c-Pro; Ala-Sar-Hyp-Pro; Ala-Asn; D-Asn-D-Ala; D-Asn;Gly, Ala;

D-Ala; β-Ala; Asn; or;

X3 is Tyr; D-Tyr; Gly, or Phe; and

R1 is H or Ac, with the proviso that X1 and X2 are not both 0; and saltsthereof.

R2 is OH or NH2

More specific compounds represented by Formula I or II above include thefollowing: G-(DBF)-Y-NH2, H-GA-Sar-Hyp-PY-NH2, H-GAG-T4c-PY-NH2,Gly-Ala-Asn-Tyr (SEQ ID NO: 2), D-Tyr-D-Asn-D-Ala-Gly, D-Tyr-D-Asn-Gly,Gly-Gly-Tyr, Gly-Ala-Tyr, D-Tyr-D-Ala-Gly, Gly-D-Asn-Tyr, Gly-βAla-Tyr,βAla-βAla-Tyr, Gly-βAla-Phe, Gly-Asn-Phe, Asn-Tyr, Ac-Gly-Tyr,Ac-Ala-Tyr, (reducedGly)-Gly-Tyr (H₂N—CH₂—CH₂—NH—CH₂—C(O)-Tyr) (SEQ IDNO: 3).

Other compounds in accordance with the present invention include: (SEQID NO: 4) gly-dab-gly-hyp-pro-tyr       |               | (SEQ ID NO: 5)gly-dapa-gly-hyp-pro-tyr        |               | (SEQ ID NO: 6)tyr-pro-hyp-gly-gln-gly |               | (SEQ ID NO: 7)tyr-pro-hyp-gly-asn-gly |               | (SEQ ID NO: 8)gly-dab-ala-gly-hyp-pro-tyr   |                  | (SEQ ID NO: 9)gly-dapa-ala-gly-hyp-pro-tyr   |                  | (SEQ ID NO: 10)tyr-pro-hyp-gly-ala-gln-gly  |                   | (SEQ ID NO: 11)tyr-pro-hyp-gly-ala-asn-gly  |                   |D-tyr-D-pro-D-hyp-gly-D-gln-gly    |                     |D-tyr-D-pro-D-hyp-gly-D-asn-gly    |                     |D-tyr-D-pro-D-hyp-gly-D-ala-D-gln-gly    |                           |D-tyr-D-pro-D-hyp-gly-D-ala-D-asn-gly;    |                           |and salts thereof.

Additionally preferred candidate compounds of the invention feature anoral bioavailability of more than about 5% as determined by anacceptable oral bioavailability assay. Generally preferred assaysinvolve intraduodenal administration of a candidate components to asuitable test subject. Availability of the compounds in a biologicalsample, preferably a blood sample, is then detected and preferablyquantified.

Further preferred candidate compounds generally satisfy Lipinski's ruleof 5. As applied, it defines bioavailability. According to the Rule,less than satisfactory adsorption is more likely when one or more of thefollowing features characterizes a particular candidate compound: 1)More than 5H-bond donors (expressed as the sum OH's and NH's), 2)Molecular weight over 500, 3) Log P is over 5, 4) More than 10H-bondacceptors (expressed as sum of N's and O's), and 5). Two parameters outof range are to be avoided for many invention embodiments.

Still further preferred compounds in accord with the invention arerelatively stable in blood plasma. An acceptable assay involvescontacting a desired candidate compound with plasma (rodent, rabbit orprimate serum, for instance), incubating the compound with the plasma,and then detecting and preferably quantifying stability of that compoundover time. Preferred plasma sources are rats, dogs, cats, mice, pigs,cows, horses, and humans. A preferred assay, sometimes referred toherein as “standard plasma stability assay” has been disclosed in thePCT application PCT/US02/05773 (WO 02/077017). Typical compounds givinggood activity in the assay feature C-terminal amidation oresterification, use of D-amino acids and derivatives of natural aminoacids, N-terminal modifications, and the cyclic structures. One or acombination of such modifications can enhance stability while retainingsubstantial biological activity.

Especially preferred hemichannel modulating compounds for use with thepresent invention include: Ac-D-Tyr-D-Pro-D-4Hyp-Gly-D-Ala-Gly-NH2(compound 1); Ac-Gly-Asn-Tyr-NH2 (compound 2); and salts thereof.

Preferred hemichannel modulating compounds exhibit significant activityin one or a combination of the standard in vitro and in vivo assaysdisclosed herein. Such compounds also have capacity to close or openhemichannels and, optionally, to modulate GJIC. Preferably, a suitablecompound enhances or reduces phosphorylation of connexin 43 (Cx43) asdetermined by the standard in vitro connexin phosphorylation assay. Amore specific assay monitors phosphorylation and/or dephosphorylation ofone or more of the tyrosine, serine and threonine amino acid residuesshown in FIG. 1. A more particular assay format follows.

-   -   1) culturing a population of about 10⁵ cells in medium such as        confluent or semi-confluent rat cardiomyocytes or H9c2 cells,    -   2) stressing the cells by washing same and subsequently        culturing them in glucose poor medium for about an hour or less,    -   3) adding compound 1 to the medium to a concentration of about        0.1 to about 200 nM for about 10 minutes to eight hours,    -   4) lysing the cells and then detecting a change in Cx 43        tyrosine, serine, and/or threonine phosphorylation relative to a        suitable control; and    -   5) measuring the change in phosphorylation as being indicative        of a compound that modulates hemichannel function.

In one embodiment of the method, the detecting step 4) further comprisestesting the candidate compound in an immunological or cell sorting-basedassay. In cases in which an immunological assay is employed that assaywill typically include contacting cells with the candidate compoundunder conditions sufficient to increase or decrease phosphorylation ofthe connexin. Preferably, the method further includes producing a lysateof the cells; and detecting increased or decreased connexinphosphorylation in the cell lysate. That increase or decrease is takento be further indicative of the hemichannel modulating compound.

Preferred immunological assays for use with the method have beendescribed and include immunoprecipitation assays, antibody captureassays, two-antibody sandwich assays, antigen capture assays,radioimmunoassays (RIAs) and the like. See generally E. Harlow and D.Lane in Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory(1988); Ausubel et al. (1989) in Current Protocols in Molecular Biology,J. Wiley & Sons, New York for a discussion relating to many standardimmunological methods, the disclosures of which are incorporated hereinby reference.

A preferred immunological assay is an ELISA assay. Thus in oneembodiment, the forgoing method further includes at least one andpreferably all of the following steps:

-   -   a) coating a solid support with a first antibody that        specifically binds the connexin, preferably connexin 43 (Cx43),    -   b) contacting the cell lysate to the solid support under        conditions conducive to forming a binding complex between the        first antibody and the connexin,    -   c) contacting the first antibody:connexin binding complex with a        second antibody, the contacting being under conditions        sufficient to form a specific binding complex between the second        antibody and any phosphorylated connexin in the first        antibody:connexin binding complex,    -   d) contacting the first antibody:connexin:second antibody        binding complex with a detectably-labeled third antibody that        binds the second antibody; and    -   e) detecting presence of the third detectably-labeled antibody        on the solid support as being further indicative of the        compound.

In one embodiment of the method, the third antibody of the method isdetectably-labeled with at least one of biotin, FITC, TRITC, radioactiveiodine or peroxidase. In another embodiment, the second antibody is ananti-phosphotyrosine antibody. In still another embodiment, the secondantibody is an anti-phosphoserine antibody. In some instances, detectionof the detectably-labeled third antibody is performed by radio- or gamascintillation counting.

In another embodiment, the immunological method employed as thedetecting step is a radioimmunoassay (RIA). Preferably, such an RIAincludes at least one and preferably all of the following steps:

-   -   a) detectably-labeling the cells before producing the cell        lysate, wherein the labeling is under conditions conducive to        labeling phosphorylated connexin, preferably connexin 43 (Cx43),    -   b) contacting the cell lysate with an antibody that forms a        specific binding complex with the connexin,    -   c) separating the binding complex from the detectably-labeled        cell lysate; and    -   d) detecting the labelled and phosphorylated connexin as being        further indicative of the gap junction modulating compound.

In one embodiment of the method, the antibody is an anti-connexinantibody. In another embodiment, the detection step further comprisesperforming a Western immunoblot assay. The RIA method is compatible witha wide range of detectable labels however for many applicationsadministration of radioactive inorganic phosphorous will be preferred.

Antibodies for use with the foregoing invention method includinganti-phosphotyrosine, anti-phosphoserine, and anti-Cx43 antibodies arecommercially available from a variety of sources such as the AmericanType Culture Collection (ATCC); Sigma Chemical Co., St. Louis, Mo.;Zymed Lab, Inc., of California; and Amersham Biosciences, U.K.

Preferred hemichannel modulating compounds identified by the foregoingstandard in vitro test show good capacity to modulate phosphorylation ofCx43, particularly at one or more of the tyrosine, serine and threonineresidues shown in FIG. 1. More preferred of such compounds exhibit atleast a 5% change in such phosphorylation (measured as an increase ofdecrease in the presence of one or more of phosphotyrosine,phosphoserine, and/or phosphothreonine on Cx43), preferably at leastabout 10%, more preferably at least about 50% change relative tostressed cells in which the compound is present at a concentration ofbetween about 0.1 nM to about 200 nM.

See Examples 2-5 below for a particular example of the standard in vitroconnexin phophorylation assay.

As discussed, the invention also features a standard in vitro uptakeassay that can be used to detect and optionally characterize hemichannelmodulating compounds. The assay can be used alone or in combination withthe standard in vitro connexin phosphorylation assay. A particularembodiment of the standard in vitro uptake assay is described asfollows.

-   -   1) culturing a population of about 10⁵ confluent rat ventricular        myocytes in medium,    -   2) stressing the cells by replacing the medium with glucose poor        medium for less than about 2 hours, preferably about 30 minutes,    -   3) adding compound 1 to the medium to a concentration of about        0.01 pM to 100 nM,    -   4) adding calcein dye to the medium to a concentration of about        10 to about 500 micromolar for less than about 2 hours and        preferably about 30 minutes,    -   5) detecting a change in uptake of the calcein into the cells;        and    -   6) measuring the change as being indicative of a compound that        modulates hemichannel function.

The step of detecting the change in calcein uptake can be performed byone or a combination of standard methods including fluorescent lightmicroscopy and/or related cell sorting methods. See Example 6 for aparticular example of the standard in vitro uptake assay.

Preferred compounds detected by the forgoing standard in vitro uptakeassay will exhibit at least about a 5% decrease in dye uptake (closecell hemichannels) relative to stressed cells, preferably at least abouta 20% decrease and more preferably at least about a 50% decrease in thepresence of between about 0.01 pM to 100 nM of the compound to betested.

As also discussed, the invention provides the in vitro cell volume assaywhich assay can be alone or in combination with one or both of thestandard in vitro connexin phosphorylation and the standard in vitrouptake assay. A particular embodiment of the standard in vitro cellvolume assay is as follows.

-   -   1) culturing a population of about 10⁵ confluent rat ventricular        myocytes in medium,    -   2) loading the cells with between from about 0.5 to about 100        micromolar calcein-AM, preferably about 5 micromolar, for less        than about 2 hours and preferably about 15 minutes    -   3) estimating the volume of the cells by detecting and        preferably quantifying signal from the calcein-AM,    -   4) adding compound 1 to the medium to a concentration of about        0.01 nM to about 100 nM,    -   5) stressing the cells by culturing in glucose poor medium,    -   6) detecting a change in cell volume relative to a suitable        control; and    -   7) measuring the change as being indicative of a compound that        modulates hemichannel function.

The step of detecting the change in cell volume can be performed by oneor a combination of standard methods including laser confocal microscopyand/or related cell sorting methods. Example 7 provides a particularexample of the standard in vitro cell volume assay.

Preferred compounds detected by the forgoing standard in vitro cellvolume assay will exhibit at least about a 5% decrease in cell volume(close hemichannels) relative to stressed cells, preferably at leastabout a 20% decrease and more preferably at least about a 50% decreasein the presence of between about 0.01 nM to 100 nM of the compound to betested. Compounds selected in accord with this assay will inhibit flowof osmolytes through hemichannels and help maintain normal cell volumeduring conditions that produces cellular swelling. Importantly, cellswelling is associated with impaired perfusion in organs surrounded by afibrous capsule (e.g., heart, kidney, skeletal muscle) or bone (brain,spinal cord) and therefore compounds with hemichannel blockingproperties may be useful in the treatment of diseases associated withcellular swelling.

Suitable control experiments are generally tailored for use in aparticular assay format. For example, most control experiments involvesubjecting a test sample (e.g, a population of cultured ratcardiomyocytes) to non-stressful conditions such as incubation inmedium. Typically, water, buffer, phosphate-buffered saline or the likeis added to the assay instead of the compound(s) to be tested inparallel or separately. A desired assay is then conducted in accord withthe present methods. Specific examples of suitable controls are providedin the Examples section.

Practice of the invention is compatible with a wide spectrum ofconventional detection assays. Such assays can be performed manually,semi-manually, or in an automated format as needed. For applications inwhich rapid or large scale screening strategies are needed, theinvention is additionally compatible with standard “high-throughput”and/or “ultra-high throughput” screening methodologies. Examples of suchmethods include immunological and cell sorting type assays such as thosedescribed herein.

As discussed, the present invention is compatible with a wide spectrumof testing strategies. Thus in some embodiments, further testing ofcandidate compounds will be performed in vivo to test for and preferablyconfirm hemichannel modifying activity. For instance, one or acombination of the standard in vitro methods described herein can beused to further test compounds for hemichannel modulating activity inthe standard in vivo arrhythmia model. The standard in vivo mousearrhythmia assay, described below as Reference Example 1, has beendisclosed in the PCT/DK01/00127 (WO 01/62775) and PCT/US02/05773 (WO02/077017) by B. Larsen et al.

Candidate compounds identified by one or more of the invention methodshave a variety of important applications including use a probes foridentifying gap junctions and particularly hemichannels in a range ofcells, tissues and organs. Also, such compounds can be used as probesfor detecting gap junctions and hemichannels in various developmentaland disease states, and as probes for detecting those structures withdistinct carbohydrate decoration and/or phosphorylation patterns. Suchcompounds find further use as solid support components for purifying gapjunction components according to standard chromatographic procedures.

Compounds selected by the in vitro and/or in vivo tests described hereincan be used as medicaments for preventing or treating conditionsimpacted by increased opening of hemichannels (increased cell membranepermeability to the extracellular compartments) eg., wounds such asburns, thromboses, respiratory and metabolic acidosis, tissue swellingeg., due to bacterial or environmental toxins, and focal arrhythmias.Other medicaments of the invention can be employed to protect cells fromdisruptive volume changes. Such changes can be facilitated by one or acombination of factors including excessive heat, ischemia, toxins,inflammation, oedema, disturbance of electrolytes, increased levels ofglucose, and diabetic late complications. Treatment of chronic atrialfibrillation associated with increased serine-phosphorylation is alsocontemplated. Additional compounds can be used as medicaments toprevent, treat, alleviate, or reduce the severity of the followingconditions. See also the PCT/DK01/00127 (WO 01/62775) and PCT/US02/05773(WO 02/077017) application.

The therapeutic methods of the invention generally compriseadministration of a therapeutically effective amount of one or acombination of the hemichannel modulating compounds disclosed herein toa subject in need of such treatment, such as a mammal, and particularlya primate such as a human. Treatment methods of the invention alsocomprise administration of an effective amount of a compound of FormulaI or II as defined above to a subject, particularly a mammal such as ahuman in need of such treatment for an indication disclosed herein.

Typical subjects include mammals suffering from one or a combination ofdisorders as provided herein including the conditions described below inSections A-R.

A. Nervous Tissue

It is well known that microglia are the main immune effector of thecentral nervous system (CNS), and that they are activated in response toa wide range of injuries that trigger brain inflammatory responses,including head injury and ischemia, neurodegenerative diseases,autoimmune diseases, infectious diseases, prion diseases, and braintumors. Activated microglia migrate to injured CNS areas, where theyproliferate and gradually remove cell debris. Eugenin et al showed thatmicroglia can communicate with each other through gap junctions that areinduced by inflammatory cytokines (Eugenin, E A, et al. Proc. Natl.Acad. Sci. USA, Vol. 98, 4190-4195, 2001). This was demonstrated in thefollowing experiments. At brain stab wounds, microglia progressivelyaccumulated over several days and formed aggregates that frequentlyshowed Cx43 immunoreactivity at interfaces between cells. In primaryculture, microglia showed low levels of Cx43 determined by Westernblotting, diffuse intracellular Cx43 immunoreactivity, and a lowincidence of dye coupling. Treatment with the immunostimulant bacteriallipopolysaccharide (LPS) or the cytokines interferon-gamma (INF-gamma)or tumor necrosis factor-alpha (TNF-alpha) one at a time did notincrease the incidence of dye coupling. However, microglia treated withINF-gamma plus LPS showed a dramatic increase in dye coupling that wasprevented by coapplication of an anti-TNF-alpha antibody, suggesting therelease and autocrine action of TNF-alpha. Treatment with INF-gamma plusTNF-alpha also greatly increased the incidence of dye coupling and theCx43 levels with translocation of Cx43 to cell-cell contacts. Thecytokine-induced dye coupling was reversibly inhibited by18-glycyrrhetinic acid, a gap junction blocker. Cultured mouse microgliaalso expressed Cx43 and developed dye coupling upon treatment withcytokines, but microglia from homozygous Cx43-deficient mice did notdevelop significant dye coupling after treatment with either INF-gammaplus LPS or INF-gamma plus TNF-alpha.

Forced expression of gap junction proteins, connexins, enables gapjunction-deficient cell lines to propagate intercellular calcium waves.Cotrina et al. demonstrated that ATP secretion from poorly coupled celllines, C6 glioma, HeLa, and U373 glioblastoma, is potentiated 5- to15-fold by connexin expression. These observations indicate thatcell-to-cell signaling associated with connexin expression results fromenhanced ATP release mediated through connexin hemichannels (Cotrina M Let al. Proc Natl Acad Sci USA 1998 Dec. 22; 95(26):15735-40; Cotrina etal.: J Neurosci 2000 Apr. 15; 20(8):2835-44). Moreover, duringconditions with metabolic stress (e.g., ischemia), hemichannel-mediatedATP release from astrocytes may affect neighboring neurons and elicitelevations in intracellular calcium with in turn may turn in apoptosisand neuronal death.

Due, for instance, to the phosphorylation of Cx43 tyrosine by compound 1and compound 2, administration of these compounds will assist inhemichannel closing and facilitate the intercellular communication ofmicroglia and thereby augment or speed up the “healing” processes in theaforementioned diseases (brain inflammatory responses, including headinjury and ischemia, neurodegenerative diseases, autoimmune diseases,infectious diseases, prion diseases, and brain tumors). Furthermore, itis expected that closing of hemichannels will prevent ATP release andthus reduce spreading of the primary injury.

B. Lung Tissue: Alveolar Cells

Alveolar intercellular communication via gap junctions between alveolarcells is important for the propagation of ion transport, mechanochemicalsignal transduction, regulation of cell growth and secretion ofsurfactant factor (Ashino Y, et al. (Am J Physiol Lung Mol Physiol 2000;279: L5-L13)). In vivo repair after acute and chronic inflammatorydamage of the alveolar region of the lung involves formation offibronectin as part of the extracellular matrix (Charash W E, et al. (AmRev Respir Dis 1993; 148: 467-476) and Torikata C, et al. (Lab Invest1985; 52: 399-408)). Alveolar epithelial cell culture studies havedemonstrated an increased number of gap junctions in parallel to anincrease of extracellular fibronectin concentration (Alford A I, RannelsD E. (Am J Physiol Lung Cell Mol Physiol 2001; 280:L680-L688)). In vivoanimal studies have found a decreased number of gap junctions afternitrogen dioxide induced severe pulmonary inflammation both in thealveolar tissue, the walls of the terminal bronchioles, alveolar ductsand peribronchiolar alveoli. These findings were dose dependent.However, if pretreated with taurin this loss of gap junctions wasprevented in parallel with less pronounced inflammatory reactions.Similar findings were seen after irradiation of rat lung and aftertreatment with the chemotherapeutic compound, bleomycin.

Thus, maintaining the gap junctional communication in lung tissue isimportant for preventing lung fibrosis and decreased amount of connexinis seen as a reaction to inflammatory processes, to various toxicstimuli, such as gas inhalation, airborne destructive substance andirradiation. Pretreatment with a compound of the invention thatphosphorylates Cx43 tyrosine residues, and facilitates hemichannelclosing and/or gap junction opening or gap junctional communication willbe indicated prior to therapeutic irradiation where the lungs areexposed, e.g. in lung cancer, treatment of breast cancer, thyroid andesophageal cancers.

Treatment with a compound that facilitates or mediates hemichannelclosing and/or gap junction opening will prevent further deteriorationof lung function in emphysema, asbestosis, silicosis, lung fibrosis,pneumonitis, drug induced lung fibrosis and in patients exposed topulmonary toxic gasses such as nitrogen dioxide. Treatment willpreferably be added on to conventional treatment of these conditions.The compound may be administered orally, parenterally, nasally, or viapulmonary inhalation.

C. Smooth Muscle

1. Vascular System

Intercellular communication through gap junction channels plays afundamental role in regulating and modulating vascular myocyte tonethroughout the vascular tree (Christ G J, et al. Circ Res. 1996; 79:631-646)). Another important role of gap junction communication is thespread of hyperpolarization among smooth muscle cells involved invascular relaxation response (Benny J L, et al. Physiol Heart CircPhysiol 1994; 266: H1465-72)).

The specialized functions of the endothelium require gap junctionintercellular communication between endothelial cells within themonolayer and between endothelium and other cells present in the vesselwall. The communication between these different cell types via gapjunctions in coronary capillaries as well as in all other vessels hasbeen documented in several studies. Evidence of involvement in adaptivearteriogenesis has also been demonstrated (Cai W-J, et al. J Mol CellCardiol 2001; 33: 957-67), Wang H-Z, et al. Am J Physiol Cell Physiol.2001; 281: C75-88), Schuster A, et al. Am J Physiol Heart Circ Physiol.2001; 280: H1088-96)).

In different vascular patophysiological situations where the endothelialmonolayer is disrupted as in diet induced hypercholestrolemic lesionsthe gap junction communication is decreased in the vascular smoothmuscles (Polacek D, et al. J Vasc Res 1997; 34: 19-30). Injury at theendothelial cellular layer is seen during venous stasis and whenthrombophlebitis is developed. Kwak B R, et al. Molec Biol Cell 2001;12: 831-845 has clearly demonstrated that gap junction communicationserves to coordinate cell migration during endothelial repair and alsoare important for capillary sprouting during angiogenesis.

Treatment with compounds of the invention that facilitate hemichannelclosing and/or gap junction communication will improve the impairedinter cellular communication in the affected vascular areas, and will beparticularly useful during organ ischemia, e.g. claudicatio intermittensand myocardial infarction.

However after balloon catheter injury in rat carotid the vascularhealing process is characterised by increased gap junctioncommunication. (Yeh H I, et al. Arterioscle Thromb Vasc Biol 1997;17:3174-84). A suitable invention compound will be administered beforethe balloon intervention and is preferably an add-on therapy toconventional medical treatment of this condition. Administration of thecompound will preferably be parenterally. Effect can be tested in tissuesampled before and at different time after the balloon catheter injury.Faster healing of the endothelial surface will be seen usingconventional microscopy. Also improvement of gap junction communicationwill be found. See also Arterioscle Thromb Vasc Biol 1997; 17:3174-84).

2. Erectile Dysfunction

In Corpus cavernosum a syncytial cellular network is established via gapjunctions and is critical to erectile function and ensures that thecorporal and arterial smooth muscle cells of the penis respond in auniform and coordinated manner. (Christ G J. (Int J Impot Res. 2000; 12suppl. 4: S15-25), Melman A, Christ J C. (Urolog Clin North America.2001; 28: 217-31)). Disturbed erectile function is seen in diabetes,arteriosclerosis, different neurological diseases and many chronicdiseases. From studies in diabetes an inverse correlation between neuralinnervation and intercellular coupling point towards the potentialfunctional plasticity of the corporal environment although notestablishing the functional intercellular communication via gapjunction.

Treatment with a compound that facilitates hemichannel closing and/orgap junction opening will improve the communication via the gap junctionand thereby normalize the complex coordination between the smooth musclecells in corpus cavernosum and the vessels.

In vivo pharmacological testing of erectile function of the compoundscan be tested 10 weeks after streptozotocin (35 mg/kg i.p.) induceddiabetes in rats (8 weeks old) as described by Rehman J, et al. (Am JPhysiol 1997; 272: H1960-71). Penile reflexes and the intracavernouspressure are measured during local and systemic administration ofdifferent doses of the different hemichannel modulating compounds withmeasures and techniques described by the same research group. Anincrease in penile reflexes and in the intracavernous pressure of 25% orabove can be seen.

Treatment of erectile dysfunction can be administered either locally inthe penil corpus, as subcutanous injection or orally. Treatment will beeither monotherapy or add-on to conventional treatment of thiscondition.

3. Incontinence

Smooth muscles in the urine bladder are characterized by phasiccontractions and show spontaneous phasic contractions. However thebladder is in the healthy condition able to contain several hundredmilliliters of urine without showing an increased intravesical pressure.In contrast to the normal bladder unstable bladders develop spontaneousincreases in intravesical pressure related to urge incontinence (TurnerW H, Brading A F. (Pharmacol Therap. 1997; 75: 77-110). Compared togastrointestinal smooth muscle, bladder smooth muscles does notspontaneously generate co-ordinated contractions (Stevens R J, et al.(Am J Physiol. 199; 2777: C448-60), Hashitani H, et al. (J Physiol.2001; 530: 273-86)). Both electrical and morphological communicationsvia gap junctions between smooth muscle cells in the bladder hasrecently been demonstrated (Hashitani H, et al. (J Physiol. 2001; 530:273-86), Wang H-Z, et al. (Urology. 2001; Suppl 6A: 111)). Theimportance of these gap junctions was demonstrated by specificinhibition of the communication. Waves of spontaneous excitation inbladder smooth muscle propagate through gap junctions.

The uncontrolled urged incontinence will therefore be regulated viatreatment with a hemichannel closing compound or a gap junction opener.Administration will be parenterally, orally or into the urinary bladder.Administration will preferably be as an add-on to treatment with drugsintended to normalize muscle contraction in the urine bladder.

Myoepithelial cells as presented in submandibular glandular ducts, inurether, in gall ducts, pancreatic ducts, tear duct are connected withgap junctions and intercellular communication is essential for thesynchronization of contractile function of the myoepithelial cells(Taugner R, Schiller A. (Cell Tissue Res. 1980; 206: 65-72). Disturbedcontractility in these ducts can be normalized by treatment with ahemichannel closing compound or a gap junction opener administeredeither parenterally or orally.

D. Healing

Prophylactic effect of treatment with a hemichannel closing agent or gapjunction opener, such as compound 1 and compound 2, can be tested in anexperimental set up as described by Yeh H I, et al. (Arterioscle ThrombVasc Biol 1997; 17:3174-84). compound 1 or compound 2 can beadministered before the balloon intervention using dosages in the rangeof 10-11 to 10-8 depending upon the compound's biological kinetics, e.g.as determined in the calciumchloride induced arrhythmia model describedabove. Tissue can be sampled before and at different time after theballoon catheter injury. Faster healing of the endothelial surface willbe seen using conventional microscopy. Administration of the compoundwill be, e.g. parenterally.

Healing progresses in a series of overlapping phases beginning withhaemostasis (coagulation). The second phase of the healing process is acascade of inflammatory responses where microphages accumulates at thewound side and formulation of granulation tissue starts involvingfibroblast and lymphocytes among other component. Epithelial cells willthen start to migrate from the border of the wound to cover the area.Capillary spouting from the normal tissue into the wound is alsoinvolved in order to ensure supply of nutrients, oxygen and thedifferent cells. All the cells and the capillary endothelium cells havean lively intercellular communication via gap junctions (Abdullah K M,et al. (Endocrine. 1999; 10: 35-41). Areas with low oxygen supply and/orhigh concentration of free radicals often seen in wounds with necrotictissue, in diabetes, in arteriosclerosis, in surgery wounds, oedema,infection, burn wounds and in venous insufficiency will lower the gapjunction communication (Nagy J I, et al. Cell Growth Diff. 1996; 7:745-51)).

Treatment with a hemichannel closing compound or a gap junction openerwill ensure maximal gap junction communication between the differentcells considered to play an important role in the complicated repairprocess and thereby improve the wound repair. The compound will beadministered topically, systemically or orally.

E. Diabetic Retinopathy

Diabetic retinopathy can be diagnosed very early after onset of thedisease by identifying alterations in the rate of blood flow (BursellS-V, et al. (Curr Eye Res. 1992; 11: 287-95), breakdown in theblood-retinal barrier (Cunha-Vaz J G, et al. (Br J Ophthalmol. 1975; 59:649-56), Do Carmo A, et al. (Exp Eye Res. 1998; 67: 569-75)) and/or lossof autoregulation (Kohner E M, Patel V, Rassam S M B. (Diabetes 1995;44: 603-607)). By using both tracer transport and double cell patchclamp techniques Oku H, et al. (Invest Ophthalmol Vis Sci. 2001; 42:1915-1920) have demonstrated an extensive cell-to-cell coupling. Aclosure of gap junction pathways disrupts the multicellular organizationof retinal microvessels and contribute to diabetic retinal vasculardysfunction. Zhou Z Y, et al. (Neuroscience. 2001; 102: 959-67) furtherdemonstrated that reactive oxygen are involved in retinal gap junctionaluncoupling and a recoupling when gluthation is supplied.

Hemichannel closers' effect on diabetic retinopathy can be studied invitro using the streptozotocin induced diabetic rat model as describedabove. Freshly isolated retinal microvessels (Sakagami K, et al. JPhysiol (Lond). 1999; 521: 637-50) will be transferred to coverslip asdescribed by Oku H, et al. (Invest Ophthalmol Vis Sci. 2001; 42:1915-1920). In this preparation the intercellular communication betweenthe cells in the vascular wall will be measured either with dye or withtracer. Different concentrations in the range of 10-10-10-7 M of thecompounds of the invention, eg., gap junction openers compound 1 orcompound 2 can be tested and a significant increase in intercellularcommunication compared to baseline will be seen in the diabetic retina.Similar improvement will be seen when compared to controls (healthyanimals). Treatment will be systemic, locally or orally. Therapy ispreferably an add-on to conventional antidiabetic treatment.

Not only diabetic retinopathy but also other vascular abnormalities inthe retina as for instance arteriosclerosis will benefit from increasedclosing of hemichannels or an improved gap junction communication bytreatment with a compound that assist Cx tyrosine phosphorylation.compound will be administered parenterally.

F. Cardiac Disorders

1. Atrial-Ventricular (AV) Blockade

Intercellular communication in the cardiac av node is maintained via gapjunctions. Decreased function lead to decreased conduction and may leadto total a-v blockade.

AV blockade is seen in acute myocardial infarction, in ischaemic heartdisease, digitalis intoxication, calcium channel blocker intoxicationand a hemichannel closing compound will improve the av conduction.Administration of hemichannel closing compound shall be eitherparenterally or orally.

2. Atrial Fibrillation

Nao et al. 2001 have found decreased serine phosphorylation of connexin40 in myocardial cells of patients suffering from chronic atrialfibrillation (AF). Decreased serine phosphorylation of connexin is knownto be connected with uncoupling of cells which may lead to AF. If thiscondition of chronic AF is also characterized in decreased tyrosinephosphorylation of connexins, then treatment with a compound such asCompound 1 or 2 herein may increase tyrosine phosphorylation, closehemichannels, open gap junction channels and remove the causes of AF.

3. Ischemia/Reperfusion Injury

During regional ischemia, hearts are exposed to metabolic stress thatcauses cell swelling which in turn increases tissue pressure and reducesperfusion of the ischemic border zone tissue. It is believed that thereduced perfusion in the ischemic border zone contributes to the gradualspreading of the infarct, which is associated with further impairment ofcardiac function. As shown in Examples 7 and 8, Compound 1 prevents cellswelling during ischemia, reduces infarct size and prevents impairmentof cardiac function after mypocardial infarction.

Moreover, coronary perfusion is reestablished in patients withmyocardial infarction by either administering a thrombolytic agent or bypercutaneous transluminal coronary angioplasty (PTCA). Althoughrestoration of blood flow is a prerequisite for myocardial salvage,reperfusion itself may lead to additional tissue injury beyond thatgenerated by ischemia alone—this is known as “reperfusion injury”.Mechanisms proposed to contribute to reperfusion injury are many,including oxygen free radical overload, neutrophil-mediated myocardialinjury, intracellular calcium overload, and changes in osmoticenvironment (Wang et al., Cardiovasc Res. 2002 July; 55(1):25-37.Review).

There is recognition that the osmolarity of bodily fluids is strictlycontrolled so that most cells do not experience changes in osmoticpressure under normal conditions, but osmotic changes can occur inpathological states such as ischemia, septic shock, and diabetic coma.The primary effect of a change in osmolarity is to acutely alter cellvolume. If the osmolarity around a cell is decreased, the cell swells,and if increased, it shrinks. In order to tolerate changes inosmolarity, cells have evolved volume regulatory mechanisms activated byosmotic challenge to normalise cell volume and maintain normal function.In the heart, osmotic stress is encountered during a period ofmyocardial ischemia when metabolites such as lactate accumulateintracellularly and to a certain degree extracellularly, and cause cellswelling. This swelling may be exacerbated further on reperfusion whenthe hyperosmotic extracellular milieu is replaced by normosmotic bloodand it may even cause rupture of the cardiac cell membranes (Wright A R,Rees S A.: Pharmacol Ther. 1998 October; 80(1):89-121. Review). Thus, itis believed that compounds selected in accord with the present inventionwill prevent myocardial damage during reperfusion, eg., by a mechanismsimilar to the effect described in Example 7 (FIG. 9A).

In addition, it has been reported that during the commonly used PTCAprocedure, the atheroschlerotic plaque is ruptured which causesmicroembolization in the microcirculation distal to the site of the PTCAprocedure, which is associated with accelerated progression of heartfailure and increased mortality Henriques J P et al:. Eur Heart J 2002July; 23(14):1112-7). As shown in Examples 7 and 8, Compound 1 preventscell swelling during ischemia, reduces infarct size and preventsimpairment of cardiac function after mypocardial infarction and theseproperties of compounds will reduce injury during reperfusion.

Accordingly, the invention provides a method of cytoprotecting tissue oran organ of a mammal in need of such treatment. In one embodiment, themethod includes administering a therapuetically effective amount of atleast one compound selected from the group consisting of the compoundsrepresented by Formula I or II. By the phrase “cytoprotecting” is meantreducing, preventing or alleviating symptoms associated with unwantedcell swelling. Particular tissues and organs that will benefit from themethod include those confined or otherwise impacted by a fiborouscapsule such as heart or kidney. Also included are tissues associatedwith bone such as brain, spinal cord and bone marrow.

In one embodiment, the method further includes exposing the tissue ororgan of the mammal to ischemic conditions such as those describedherein. An example is heart infarction (heart attack) in which theischemia is associated with harmful myocardial cell swelling. In oneembodiment, such swelling can be reduced or avoided by administering atleast one of Ac-D-Tyr-D-Pro-D-4Hyp-Gly-D-Ala-Gly-NH2 (Compound 1) andAc-Gly-Asn-Tyr-NH2 (Compound 2) to the mammal.

As discussed a method of preventing or treating reperfusion injury in amammal is also provided by the invention. In one embodiment, the methodincludes administering a therapuetically effective amount of at leastone compound selected from the group consisting of the compoundsrepresented by Formula I or II. In a more specific embodiment, themethod further includes the heart of the mammal to infarct conditions.According to the method, it is desirable to establish coronaryperfusion, typically as quickly as possible. In some embodiments, themethod will further include administering a thrombolytic agent (eg.,tissue plasminogen activator (“TPA”)) or providing coronary angioplastyto facilitate coronary perfusion into the infarcted heart. In oneembodiment, the compound is at least one ofAc-D-Tyr-D-Pro-D-4Hyp-Gly-D-Ala-Gly-NH2 (Compound 1) andAc-Gly-Asn-Tyr-NH2 (Compound 2).

G. Immunology: Cell Maturation

Cell-to-cell interactions are crucial for lymphocyte maturation andactivation. A wide rage of membrane molecules ensure intercellularadhesion and enabling cell-cell signalling during cell migration andactivation in the immune system. Circulating human T, B and NKlymphocytes express Cx43 and active gap junctions between the cells havebeen demonstrated using dye methods as described previously. It has alsobeen demonstrated that decrease in intercellular gap junctional couplingmarkedly decrease the secretion of IgM, IgG and IgA indicating thatintercellular signaling across gap junctions is an important componentof the mechanisms underlying metabolic cooperation in the immune system(Oviedo-Orta E, et al. (Immunology. 2000; 99: 578-90), Oviedo-Orta E, etal. (FASEB. 2001; 15:768-774)).

In subchronic or chronic inflammation a local increase in synthesis ofimmunglobulins is desirable independent of aethiology. Duringinflammation the tissue is often different from the normal healthytissue and low oxygen tension produces uncoupling of the intercellulargap junctional communication. The importance of low oxygen for GJICuncoupling has been demonstrated in several different cell systemssuggesting that oxygen tension is a universal regulator of GJIC. Saiduncoupling is a result of altered tyrosine and/or serine and/orthreonine phosphorylation of connexins in gap junction channels, andadministration of a compound of the invention such as compound 1 or 2herein will counteract this effect, close hemichannels and restore GJIC.

H. Improving GJIC

There are basically two ways of maintaining GJIC, either by keeping gapjunction channels open or by facilitation of docking of hemichannels. Inthe treatment or prevention of disease states characterized in presenceof ischemic conditions, both ways are preferred or recommended.

In primary cultures of neonatal rat ventricular cardiomyocytes,deprivation of oxygen and glucose leads to a decrease in thenoradrenalin-induced stimulation of phosphoinositol (PI) turnover toapp. 50% of the level at normal atmospheric and nutritional conditions.The gap junction modifier compound 1 has been shown to normalise thisimpaired noradrenalin-induced stimulation of PI turnover during oxygenand glucose deprivation by raising PI turnover to app. 90% of the normallevel. Moreover is has been shown that compound 1 does not alter thenoradrenalin-induced level of PI turnover during normal atmospheric andnutritional conditions (Meier, E and Beck, M M: 2001 International GapJunction Conference, Aug. 4-9, 2001, Hawaii, USA, abstract no. 132).Likewise, in cultured human osteoblast cultures and in osteoblastic ratosteosarcoma cell lines hypoxia decreased intracellular calcium wavepropagation as measured as dye transfer after Lucifer Yellow injections.This decrease could be completely reversed by treatment with compound 1(Teilmann, S C, et al.: 2001 International Gap Junction Conference, Aug.4-9, 2001, Hawaii, USA, abstract no. 176).

Due to cellular uncoupling during inflammation, for instance, a compoundthat closes hemichannels and opens gap junctions will improve synthesisof immunglobulins during inflammation.

I. Peripheral Neuropathy and Neuropathic Pain

Peripheral neuropathy and pain as seen in diabetes, during dialysis,liver cirrhosis and many other conditions are reported to involve bothsomatic and autonomic nerves. The exact mechanisms of the peripheralnerve injury in the various conditions are under investigation but nerveterminal destruction, decreased conductance, demyelination and increasedinflammatory response have been described. Common for the variousconditions in experimental set up are that increased free radicals,increased nitric oxide, oxygen stress and lack of free radicalscavengers are seen and reduction of gap junction communication isrecorded (Pitre D A, et al. (Neurosci Lett. 2001; 303: 67-71), Bolanos JP, Medina J M. (J Neurochem. 1996; 66: 2019-9), Low P A, Nickander, K K.(Diabetes. 1991; 40: 873-7), Levy D, et al. (Neurosci Lett. 1999; 260:207-9), Bruzzone R, Ressot C. J Eur Neurosci. 1997; 9: 1-6)). Thus, acompound of the invention that assists hemichannel closing byphosphorylating hemichannel connexins will be beneficial in thetreatment of peripheral neuropathy. Administration will be parenterally.

J. Hearing Deficit

Noise induced hearing loss, presbycusis known to be associated withproduction of free radicals are related to inhibition of gap junctioncoupling between both Hensen cells and Deiters cells from Corti's organin the cochlea (Todt I, et al. (J Membrane Biol. 2001; 181: 107-114),Blasits S, et al. (Phlugers Arch. 2000; 440: 710-12) Lagostena L, et al.(J Physiol. 2001; 531: 693-707)). The gap junction communication betweenthese supporting cochlear cells provides the important homeostasis forthe sensory cells and thereby a normal neuronal activity of outer haircells (Johnstone B M, et al. (J Physiol 1989; 408: 77-92)). Thiscommunication is disrupted during oxidative stress (Todt I, et al. (JMembrane Biol. 2001; 181: 107-114). Acquired or age dependent hearingloss will be prevented when treated with a compound which can increasephosphorylation of tyrosine residues in connexin hemichannels (close thehemichannels) and maintain gap junction communication in the supportivecells. A suitable compound of the invention can be administeredparenterally.

Melanocytes in the vestibular organ dark cell area are communicatingheavily via gap junction and may play a role in transporting materialbetween the endolymph and perilymph and also be of importance inmaintaining the homeostasis of the microenvironment in the inner ear(Masuda M, et al. (Anat Rec. 2001; 262; 137-146)). Endolymphatic hydropsis related to various clinical conditions characterized by dizziness andreduced hearing. A decreased capacity of gap junction communication maybe of importance in regulating transmembrane transport of severalsubstances originally secreted or excreted via specific types oftransporters.

K. Age Dependent Anemia and Bone Marrow Transplantation

Existence of functional gap junctions between haematopoietic progenitorcells and stromal cells of the haematopoietic microenvironment was manyyears controversial but studies have now proofed the existence of humangap junction communication (Rosendaal M, et al. Tissue Cell. 1991; 23:457-470), Dürig J, et al. (Brit J Haematol. 2000; 111: 416-25)). It hasalso been demonstrated that the communication is bi-directional favoringthe hypothesis that stromal cells control the proliferative behaviour ofthe haematopoietic progenitor cells, but also their functional statuscan be regulated by immature haematopoietic cells (Gupta P, et al.(Blood. 1998; 91: 3724-3733)).

With age the functionality of the haematopoietic tissue is decreased andanemia is often seen in elderly people. Reduced capacity ofhaematopoietic tissue is also seen in haematological malignancies andafter treatment with chemotherapeutics. Bone marrow transplantation fromdonor is used to prevent pancytopenia.

The effect of a compound of the invention that assists in hemichannelclosing and/or facilitates gap junction communication will be studied inpretreated rats exposed to high dose cyclophosphamide. In these animalsthe bone marrow has stopped producing mature haematopoietic cells.Number of reticulocytes at different time intervals aftercyclophosphamide will be significantly higher in the animals pretreatedwith the connexin tyrosine phosphorylating compound 1 using doses ofabout 100 μL of 10⁻¹⁰ M to about 10⁻⁸ M compound 1 compared tonon-pretreated animals. Administration of suitable compounds of theinvention will be parenterally.

L. Pituitary and Hypothalamic Hypofunction

Hormones from the anterior pituitary gland show circadian variation insecretion within minutes, hours, days and seasons. The part of thenervous system responsible for most circadian rhythm is localized to apair of structures in the hypothalamus known as the suprachiasmaticnucleus. In this center this biological clock is intrinsic in theindividual cells. However coordinated electrical activity is mediated toneighboring cells via gap junction communication. (Colwell C S. (JNeurobiol. 2000; 43: 379-88)). Because also the anterior pituitary lacksdirect innervations, gap junction-mediated cell-to-cell communicationwithin the gland must be indispensable for the adequate cell-to-cellcoordination and synchronization required to ensure appropriate andtimed hormone secretion. (Vitale M L, et al. (Biol Reporo. 2001; 64:625-633)). Guerineau N C, et al. (J Biol. Chem. 1998; 273: 10389-95)concluded that spontaneously active endocrine cells are either singleunits or arranged in synchronized gap junction-coupled assembliesscattered throughout the anterior pituitary gland. Synchrony betweenspontaneously excitable cells may help shape the patterns of basalsecretion. From the anterior pituitary gland, growth hormone, prolactin,adrenocortical hormone, thyroid hormone, and gonadotropin hormones aresynthesized under control from hypothalamus stimulating hormones. One ofthe mechanism in dysrhythm of the complicatedhypothalamic-pituitary-endocrine glands within one of the axis istherefore also related to reduced communication via gap junctions. Thediseases are diabetes insipidus, hypogonadotrope hypogonadism,myxoedema, adrenocorticoid hypofunction, and dwarfism. Treatment with asuitable compound of the invention, preferably a gap junction opener,can improve the symptoms.

Also the neurons in the suprachiasmatic nucleus of the hypothalamus aredependent on optimal gap junction communication. In the axis mentionedabove gap junction opener with mode of action in this region will alsobenefit patients with disturbed circadian rhythm (Shinohara K, et al.(Neusosci Lett. 2000; 286: 107-10).

M. Renovascular Hypertension and Nephrotoxicity

Kidney and endothelial specific gap junctions are widely distributed inthe kidney found in glomeruli, tubulus and vasculature includingintraglomerular capillaries and juxaglomerular arterioles (HaefligerJ-A, et al. (Kidney Int. 2001; 60: 190-201)). In that study the authorsdemonstrated the presence of gap junctions connecting renin-secretingcells of the afferent arteriole. The role of gap junction mightcontribute to the detection and propagation of blood borne signals, suchas those elicited by increased blood pressure. Within the kidney, suchsignals need to be converted into autocrine, paracrine and endocrinestimuli by the endothelial cells of the afferent arteriole and thetransmitted to the renin-secreting cells. Gap junction communicationplays thus an important role in forming the interconnectedjuxtaglomerular apparatus. The rapid open to close transitions of gapjunctions channels further imply a readily response to local vascularchanges ensuring the continuous feedback required to match glomerularand tubular function as well as renin secretion to physiologicaldemands. Diseases characterized by impaired renal gap junctioncommunication will benefit from treatment with compound of theinvention, preferably a specific gap junction opener, eitheradministered orally or parenterally.

Heavy metals are nephrotoxic and causes renal injury. It has beendemonstrated that the toxic metals cadmium (Fukumoto M, et al. (LifeSciences. 2001; 69:247-54)) as well as mercury (Yoshida M, et al. (ArchToxicol. 1998; 72: 192-96)) in primary cell cultures from rat proximaltubulus uncouple gap junctions and both groups suggest that renaldysfunction is related to the reduced intercellular communication.Treatment of heavy metal poisoning with a connexin tyrosinephosphorylating compound will reduce the tissue damage and prevent theprogressive tissue devastation.

An in vitro test can be performed in cell cultures from tubulus cellsand the compounds (compound 1 or compound 2 in a concentration of about10⁻¹⁰-10⁻⁷ M) prevention of gap junction uncoupling when exposed toheavy metals will be investigated. Gap junction communication will betested with Lucifer dye method as described previously.

After systemic administration of heavy metal to experimental animals(rats) renal function will be measured using ³H-insulin as a clearancemarker for glomerular filtration rate, ¹⁴C-labelled tetraethylammoniumas a clearance marker for renal plasma flow and lithium as a marker forproximal tubular function (Petersen J S, et al. J. Pharmacol. Esp. Ther.1991, 258:1-7) before and after different time of chronic treatment withheavy metals. Chronic treatment with a specific compound of theinvention, such as compound 1, will be initiated when renal function iscompromised and an significant improvement of renal function parameters(glomerular filtration rate and blood pressure) will be seen followingthe treatment. Administration of a suitable invention compound will beparenterally.

Non-infectious inflammation as well as infections with differentmicrobes induces significant non specific chronic changes in renalfunction also characterized by reduced glomerular filtration rate,decreased excretion of electrolytes and water and changes in bloodpressure. Some of these symptoms will as well be treated with a specificconnexin tyrosine phosphorylating compound and the symptoms willdecline.

N. Developing and Remodelling of Teeth

Murakami S and Muramatsu T (Anat Embryol. 2001; 203: 367-374) confirmedprevious studies that gap junction communication exists betweenodontoblasts and that cellular activity is coordinated via theseintercellular bridges (Iguchi Y, et al. (Arch Oral Biol. 1984; 29:489-497)) but in their recent study they also demonstrated that thesegap junction communications are present during the early development ofteeth (pre-odontoblast) as well as in the odontoblasts in young and oldodontoblast. Also the pulp cells subjacent to odotoblasts have gapjunctions. These findings indicate that intercellular gap junctioncommunication is important both during development of the teeth andduring lifetime when teeth are remodeled or wormed.

Treatment with a connexin phosphorylating compound of the invention,such as compound 2 which can be tested in vitro for effect onodontoblast intercellular communication in an assay which is essentiallycomparable to the osteoblast assays described herein, will normalizedisturbed development of teeth. Treatment will also facilitateremodeling of teeth and make the teeth more resistant to caries.

O. Stem Cells

Lumelsky et al (2001) have generated cells expressing insulin and otherpancreatic endocrine hormones from mouse embryonic stem cells. See NadyaLumelsky et al. in Differentiation of Embryonic Stem Cells toInsulin-Secreting Structures Similar to Pancreatic Islets. Science, 292,1389-1394, 2001). The cells self-assemble to form three-dimensionalclusters similar in topology to normal pancreatic islets wherepancreatic cell types are in close association with neurons. Glucosetriggers insulin release from these cell clusters by mechanisms similarto those employed in vivo. When injected into diabetic mice, theinsulin-producing cells undergo rapid vascularization and maintain aclustered, islet-like organization.

In the clinical context, this embryonic stem cell-based system willallow simultaneous generation and assembly of insulin-secreting andother islet cell types known to play important role in regulation ofinsulin secretion into functional structural units. These units canprovide material to optimize insulin production and analyze the finecontrol of glucose homeostasis. embryonic stem cells are ideal for thesestudies because genetic tools can be used to define the molecular basisof islet development and function. Potential for cell-based therapies isclearly an attractive goal for applications involving human and nonhumanembryonic stem and embryonic germ cells. Adult tissue may also be auseful source of functional pancreatic cells. The differentiation systemdescribed here may provide a source of functional pancreatic islets fortreatment of diabetes. This is the first report showing that the severalcell types of endocrine pancreas can be generated from embryonic stemcells in vitro. Although pancreatic islets obtained from cadavers canfunction in the liver after grafting, issues of tissue rejection andavailability remain to be resolved. It is clear that engineering ofembryonic stem cells to produce an abundant source of immunocompatibletissue for transplantation holds a growing promise for surmounting thisand other problems associated with diabetes.

Myocardial infarction leads to loss of tissue and impairment of cardiacperformance. The remaining myocytes are unable to reconstitute thenecrotic tissue, and the post-infarcted heart deteriorates with time.Injury to a target organ is sensed by distant stem cells, which migrateto the site of damage and undergo alternate stem cell differentiation;these events promote structural and functional repair. This high degreeof stem cell plasticity prompted Orlic et al (Nature 410, 701-705(2001)) to test whether dead myocardium could be restored bytransplanting bone marrow cells in infarcted mice. They sortedlineage-negative (Lin-) bone marrow cells from transgenic miceexpressing enhanced green fluorescent protein by fluorescence-activatedcell sorting on the basis of c-kit expression. Shortly after coronaryligation, Lin-c-kitPOS cells were injected in the contracting wallbordering the infarct. They found that newly formed myocardium occupied68% of the infarcted portion of the ventricle 9 days after transplantingthe bone marrow cells. The developing tissue comprised proliferatingmyocytes and vascular structures. Their studies indicate that locallydelivered bone marrow cells can generate de novo myocardium,ameliorating the outcome of coronary artery disease.

To characterize further the properties of these myocytes, theydetermined the expression of connexin 43. This protein is responsiblefor intercellular connections and electrical coupling through thegeneration of plasma-membrane channels between myocytes; connexin 43 wasapparent in the cell cytoplasm and at the surface of closely aligneddifferentiating cells. These results were consistent with the expectedfunctional competence of the heart muscle phenotype.

Since functional cells are generated from embryonic stem cells, andsince connexins are indeed expressed in these cells in infarcted hearttissue, it is believed that this will be the case for other cellsdifferentiated from embryonic stem cells. Since connexins play adominating role in the function of these tissues (including pancreaticbeta cells and heart muscle cells). compounds of the invention such ascompound 1 and compound 2 that increase connexin tyrosinephosphorylation and hemichannel closing (resulting also in increased gapjunction communication) will enhance the proliferation of embryonic stemcells into functional cells in organs wherein stem cells have beenimplanted.

Thus it is an object of the invention to provide connexin tyrosinephosphorylating compounds such as compound 1 and compound 2 to stimulatethe transition of stem cells to functional cells in transplanted organslike pancreas for treatment of diabetes mellitus, heart for treatment ofheart infarction, and basal ganglia of the brain for treatment ofParkinsons disease. In general, it is believed that these compounds willaccelerate diffentiation of stem cells, which may accelerate healingprocesses in all organs such as heart, brain, skin, bone, pancreas,liver, and other internal organs. Experiments can be performed using thegeneral experimental design with myocardial infarction as describedabove by Orlic et al, supra, with administration of compound 1 andcompound 2, for instance, repeatedly during the proliferation process.These experiments are believed to show an increase in connexin 43expression by compound 1 and compound 2 or a faster regenerativeprocess.

P. Cancer

1. Tumor Progression

During tumorigenesis, the interruption of the physiological interactionof normal cells with their neighboring cells, and loss of features ofdifferentiation are a common denominator in tumor progression.Alteration in gap junction communication is believed to be among theearliest changes during cell tumorgenesis (Wolburg H, Rohlmann A. IntRev Cytol. 1995; 157: 315-73), Klaunig J E, Ruch R J. 1990; 135-46)).

Kyung-Sun Kang, et al. (Cancer Letters 166 (2001) 147-153) have shownthat pre- and co-incubation with GeO2 in TPA treated rat liverepithelial cells abolished down-regulation of GJIC by TPA suggestingthat a substance that recovers the inhibition of GJIC may be used in theprevention or inhibition of tumor promotion. Suzuki J, Na H-K, et al.(Nutrition and Cancer, vol 36 No. 1 p. 122-8) have shown that the foodadditive lambda-carrageenan inhibits GJIC in rat liver epithelial cellssimilar to that of the well-documented tumor promotor phorbol ester(TPA), and therefore could play a role in carcinogenesis as a tumorpromoting agent. Thus, the compounds of the present invention may beused in the prevention or treatment of cancer caused by tumor promotingagents, such as TPA and lambda-carrageenan.

2. Drug Sensitivity Resistance

Increased gap junction communication improves the microenvironment intumors. See Chen et al.: Chem Biol Interact 1998 Apr. 24; 111-112:263-75

3. Metastasis

Loss of intercellular gap junction communication is associated with highmetastatic potential in all cancers with metastatic potentials.(Saunders M M, et al. Cancer Res. 2001; 61: 1765-1767), Nicolson G I, etal. Proc. Natl. Acad Sci USA. 1988; 85: 473-6)). Prevention ofmetastasis is established by treatment with a connexin tyrosinephosphorylating compound which will assist in preserving the gapjunction communication in tumors. Treatment is an add on to conventionalchemotherapy.

It is believed that administration of one or a combination of theinvention compounds in a therapeutically effective amount (eg., compound1 or compound 2) will be able to prevent or treat cancer.

Q. Miscellaneous Cells

Gap junctions also play an important role in intercellularcommunication, proliferation and differentiation in gastric mucosalcell. Gap junction opener will stimulate regenerative processes afterinduced injury (Endo K, et al. (J Gastroenterol Hepatol. 1995; 10:589-94)).

The cytoarchitecture of meniscal cells partly depends on gap junctioncommunication. The fibrocartilage part of the meniscal as well as thefibrocartilage structure of tendons depends on intercellularcommunication. During injuries gap junction openers will improve thespeed of repair.

R. Epilepsy

It may be desirable to treat disease states such as epilepsiacharacterized in abnormally high GJIC with a hemichannel opener,preferably as an adjuvant therapy in order to allow uptake of drugs orsmall regulatory molecules that serve to uncouple gap junction channels.

It is an object of the present invention to provide methods to treat orprevent one or more of the medical indications or conditions describedherein. Typically, but not exclusively, such methods will includeadministration of at least one of the foregoing compounds, preferablyone of same, in an amount sufficient to treat, prevent, or reduce theseverity of the indication or condition. Preferred compounds have beenselected by one or a combination of the in vitro and in vivo assaysdescribed herein. Particular administration strategies will be apparentof those of skill in this field and will vary depending eg., on the sex,weight, general health and specific indication or condition to betreated or prevented. As discussed, the compounds disclosed herein canbe employed as the sole active agent in invention methods.Alternatively, they can be used in “add-on” therapies such as those inwhich use of the compounds in conjunction with a recognized treatmentmethod is indicated. Preferred indications or conditions to be treatedor prevented in accord with the invention are generally associated withimpaired cellular communication or impaired gap junction function. Morespecific indications and conditions relating to the invention have beendiscussed above.

Treatment methods in accord with the invention can employ one or more ofthe compounds disclosed herein as the sole active agent. Preferably, oneof the compounds will be employed. If desired, such compounds can beused prophylactically ie., to prevent or reduce the severity of aparticular indication or condition. Alternatively, the compounds can beused in conjunction with a recognized therapeutic approach. As anillustration in embodiments in which irradiation is treated, it isgenerally preferred that the treatment method be “add on” ie., inconjunction with a recognized therapy for treating the condition. Such“add on” treatment methods of the invention can be conducted at the sametime or at a different time then the recognized therapy as needed.Established therapeutic approaches for a variety of diseases and medicalconditions have been described. See generally, Harrison's Principles ofInternal Medicine (1991) 12 ed., McGraw-Hill, Inc. and ThePharmacological Basis of Therapeutics (1996) Goodman, Louis S. 9th edPergammon Press, for example; the disclosures of which are incorporatedherein by reference.

The concentration of one or more treatment compounds in a therapeuticcomposition will vary depending upon a number of factors, including thedosage of the hemichannel modulating compound to be administered, thechemical characteristics (e.g., hydrophobicity) of the compositionemployed, and the intended mode and route of administration. In generalterms, one or more than one of the hemichannel modulating compounds maybe provided in an aqueous physiological buffer solution containing about0.1 to 10% w/v of a compound for parenteral administration.

It will be appreciated that the actual preferred amounts of activecompounds used in a given therapy will vary according to e.g. thespecific compound being utilized, the particular composition formulated,the mode of administration and characteristics of the subject, e.g. thespecies, sex, weight, general health and age of the subject. Optimaladministration rates for a given protocol of administration can bereadily ascertained by those skilled in the art using conventionaldosage determination tests conducted with regard to the foregoingguidelines. Suitable dose ranges may include from about 1 μg/kg to about100 mg/kg of body weight per day (“therapeutically effective amount”).

Therapeutic compounds of the invention are suitably administered in aprotonated and water-soluble form, e.g., as a pharmaceuticallyacceptable salt, typically an acid addition salt such as an inorganicacid addition salt, e.g., a hydrochloride, sulfate, or phosphate salt,or as an organic acid addition salt such as an acetate, maleate,fumarate, tartrate, or citrate salt. Pharmaceutically acceptable saltsof therapeutic compounds of the invention also can include metal salts,particularly alkali metal salts such as a sodium salt or potassium salt;alkaline earth metal salts such as a magnesium or calcium salt; ammoniumsalts such an ammonium or tetramethyl ammonium salt; or an amino acidaddition salts such as a lysine, glycine, or phenylalanine salt.Additionally suitable salts are provided below.

More particular hemichannel modulating compounds of the invention areused in the form of a pharmaceutically acceptable salt, an alkyl ester,an amide, an alkylamide, a dialkylamide or a hydrazide formed with theC-terminal carboxylic acid function of a linear compound or a freecarboxylic acid function, if present, of a cyclic compound. Amides andlower alkyl amides of linear compounds are among the preferred compoundsof the invention. Salts include pharmaceutically acceptable salts, suchas acid addition salts and basic salts. Examples of acid addition saltshave already been described and include hydrochloride salts, sodiumsalts, calcium salts, potassium salts, etc. Examples of basic salts aresalts where the cation is selected from alkali metals, such as sodiumand potassium, alkaline earth metals, such as calcium, and ammonium ions⁺N(R³)₃ (R⁴), where R³ and R⁴ independently designate optionallysubstituted C₁₋₆-alkyl, optionally substituted C₂₋₆-alkenyl, optionallysubstituted aryl, or optionally substituted heteroaryl. Other examplesof pharmaceutically acceptable salts are; e.g., those described inRemington's Pharmaceutical Sciences 17. Ed. Alfonso R. Gennaro (Ed.),Mark Publishing Company, Easton, Pa., U.S.A., 1985 and more recenteditions, and in Encyclopedia of Pharmaceutical Technology.

In the therapeutic methods of the invention, a treatment compound can beadministered to a subject in any of several ways. For example, ahemichannel modulating compound selected in one or a combination of thein vitro and/or in vivo assays provided can be administered as aprophylactic to prevent the onset of or reduce the severity of atargeted condition. Alternatively, the compound can be administeredduring the course of a targeted condition.

A treatment compound can be administered to a subject, either alone orin combination with one or more therapeutic agents, as a pharmaceuticalcomposition in mixture with conventional excipient, i.e.pharmaceutically acceptable organic or inorganic carrier substancessuitable for parenteral, enteral or intranasal application which do notdeleteriously react with the active compounds and are not deleterious tothe recipient thereof. Suitable pharmaceutically acceptable carriersinclude but are not limited to water, salt solutions, alcohol, vegetableoils, polyethylene glycols, gelatin, lactose, amylose, magnesiumstearate, talc, silicic acid, viscous paraffin, perfume oil, fatty acidmonoglycerides and diglycerides, petroethral fatty acid esters,hydroxymethyl-cellulose, polyvinylpyrrolidone, etc. The pharmaceuticalpreparations can be sterilized and if desired mixed with auxiliaryagents, e.g., lubricants, preservatives, stabilizers, wetting agents,emulsifiers, salts for influencing osmotic pressure, buffers, colorings,flavorings and/or aromatic substances and the like which do notdeleteriously react with the active compounds.

Such compositions may be prepared for use in parenteral administration,particularly in the form of liquid solutions or suspensions; for oraladministration, particularly in the form of tablets or capsules;intranasally, particularly in the form of powders, nasal drops, oraerosols; vaginally; topically e.g. in the form of a cream; rectallye.g. as a suppository; etc.

The pharmaceutical agents may be conveniently administered in unitdosage form and may be prepared by any of the methods well known in thepharmaceutical arts, e.g., as described in Remington's PharmaceuticalSciences (Mack Pub. Co., Easton, Pa., 1980). Formulations for parenteraladministration may contain as common excipients such as sterile water orsaline, polyalkylene glycols such as polyethylene glycol, oils ofvegetable origin, hydrogenated naphthalenes and the like. In particular,biocompatible, biodegradable lactide polymer, lactide/glycolidecopolymer, or polyoxyethylene-polyoxypropylene copolymers may be usefulexcipients to control the release of certain hemichannel modulatingcompounds of the invention.

Other potentially useful parenteral delivery systems includeethylene-vinyl acetate copolymer particles, osmotic pumps, implantableinfusion systems, and liposomes. Formulations for inhalationadministration contain as excipients, for example, lactose, or may beaqueous solutions containing, for example, polyoxyethylene-9-laurylether, glycocholate and deoxycholate, or oily solutions foradministration in the form of nasal drops, or as a gel to be appliedintranasally. Formulations for parenteral administration may alsoinclude glycocholate for buccal administration, methoxysalicylate forrectal administration, or citric acid for vaginal administration. Otherdelivery systems will administer the therapeutic agent(s) directly at asurgical site, e.g. after balloon angioplasty a hemichannel modulatingcompound may be administered by use of stents.

Abbreviations and Formulae:

Throughout the description and claims the three letter code for naturalamino acids is used as well as generally accepted three letter codes forother α-amino acids, such as Sarcosin (Sar), α-Amino-iso-butanoic acid(Aib), Naphthylalanine (Nal) including 1-naphthylalanine (1Nal) and2-naphthylalanine (2Nal), Phenylglycine Phg, 2,4-Diaminobutanoic acid(Dab), 2,3-Diaminopropanoic acid (Dapa), and Hydroxyproline (Hyp). Wherenothing is specified Hyp represents 4-hydroxyproline. The natural oressential amino acids are the amino acid constituents of proteins. Thearomatic amino acids are Phe, Tyr, Trp, 1Nal, 2Nal and His. Where the Lor D form has not been specified it is to be understood that the aminoacid in question has the natural L form, cf. Pure & Appl. Chem. Vol.56(5) pp 595-624 (1984). Where nothing is specified it is to beunderstood that the C-terminal amino acid of a compound of the inventionexists as the free carboxylic acid, this may also be specified as “—OH”.The C-terminal amino acid of a compound of the invention may be shown tohave the terminal function “—OH/NH₂” which means that there are twopreferred forms of the compound: the free carboxylic acid and theamidated derivative.

The following specific definitions apply unless otherwise specified:ASAL refers to 4-azidosalicyloyl radical; AB refers to 4-azidobenzoylradical; Fmoc refers to 9-fluorenylmethyloxycarbonyl radical; Ac refersto acetyl radical. Throughout the present disclosure unless otherwisespecified the following definitions apply; Acm refers to acetamidomethylradical; T4c refers to L-thiazolidin-4-carboxylic acid radical; Pcrefers to L-pipecolic acid radical; DNP refers to dinitrophenyl; DBF isdefined as 2-aminoethyl-6-dibenzofuranpropionic acid; Acm refers toacetamidomethyl; and Sar refers to sarcosinyl radical; DNP functions isa hapten for antibody recognition, and compounds of the invention thatcontain a DNP moiety may be preferably used as research tools.

See also PCT/DK01/00127 (WO 01/62775) and PCT/US02/05773 (WO 02/077017)for additional information.

All references disclosed herein are incorporated by reference. Thefollowing Examples are illustrative of the invention.

REFERENCE EXAMPLE 1 Standard In Vivo Mouse Arrythmia Assay

It is possible to test for antiarrythmic effects by performing acalcium-induced arhythmia model in mice.

Briefly, the antiarrhythmic effects of compounds can be tested in an invivo model of calcium-induced arrythmias according to the model of J. J.Lynch, R. G. et al. J Cardiovasc. Pharmacol. 1981, 3 49-60. Mice (25-30g) were anaesthetised with a neurolept anaesthetic combination (Hypnorm®(fentanyl citrate 0.315 mg/ml and fuanisone 10 mg/ml)+midazolam (5mg/ml)). Commercial solutions of hypnorm and midazolam were diluted 1:1in distilled water and one part diluted Hypnorm® is mixed with one partdiluted midazolam.

The anaesthesia was induced by s.c. administration in a dose of0.05-0.075 μl/10 gram mouse. An i.v. cannula was inserted into the tailvein. The lead II ECG signal was recorded continuously by positioning ofa stainless steel ECG electrodes on the right forelimb and on the lefthind limb. The ground electrode was placed on the right hind limb. Thesignal was amplified (×5.000-10.000) and filtered (0.1-150 Hz) via aHugo Sachs Electronic model 689 ECG module. The analogue signal wasdigitised via a 12 bit data acquisition board (Data Translation modelDT321) and sampled at 1000 Hz using the Notocord HEM 3.1 software forWindows NT. After a 10-min equilibration period, the test sample of drugwas injected into the tail vein. Mice pre-treated with vehicle weretested as a measure of the control level in untreated animals. Theinjection volume was 100 μl in all experiments. Infusion of CaCl₂ (30mg/ml, 0.1 ml/min≈100 mg/kg/min (calciumchlorid-2-hydrat, Riedel-deHaën, Germany)) was started 3 min after i.v. administration of drug orvehicle. The time lag to onset of 2nd degree AV-block was determined asthe time from the start of CaCl₂ infusion until the first arrhythmicevent occurred. An event of 2nd degree AV-block was defined asintermittent failure of the AV conduction characterised by a P-wavewithout the concomitant QRS complex.

Responses were expressed relative to the time until 2nd degree AV-blockoccurred in vehicle treated mice. Maximal effect of each of the testedsubstances has been summarized.

More particular methods for detecting candidate compounds with good gapjunction modifying activity further include selecting candidatecompounds that prolong the time until onset of the induced atrialventricular (AV) block by at least about 20% (score at least 2) in thestandard in vivo mouse arrhythmia assay. More preferred compoundsexhibit a prolongation of at least about 60% of the time until onset ofthe induced AV block (score at least 3) in the assay.

The following examples provide a functional assay that shows the abilityof compound 1 to both open gap junction channels and close hemichannelsin rat neonate cardiomyocytes. Thus, it is possible to obtaindifferentiated regulation of GJIC by tyrosine phosphorylation of bothhemichannels and gap junction channels.

EXAMPLE 1 Antiarrhythmic Compounds

compounds used in the invention includeAc-D-Tyr-D-Pro-D-4Hyp-Gly-D-Ala-Gly-NH2 (compound 1) andAc-Gly-Asn-Tyr-NH2 (compound 2). The compounds are synthesised accordingto standard solid phase synthesis as described in WO01/62775. A furtherexample of compounds useful in the invention is trans-resveratrol andCAPE (caffeic acid phenethyl ester). Methods for making the compoundshave been described. See e.g, the PCT/DK01/00127 (WO 01/62775) andPCT/US02/05773 (WO 02/077017) applications.

EXAMPLE 2 Analysis of Metabolic Stress Induced by Glucose Removal

Cardiac arrhythmias can be caused by disturbances of both formation andconduction of the action potential. The propagation of an actionpotential from cell to cell is mediated by the intercellular gapjunctions. These gap junctions consist of channels, which are built byspecialised proteins called connexins. There are several lines ofevidence showing that a reduced expression and/or a disturbeddistribution of the connexins, can be deleterious for normal impulsepropagation and thereby arrhythmogenic. One example is the alterationsseen during and after ischemia. During acute ischemia uncoupling of thecells are mainly believed to be caused by intracellular acidification,increased intracellular Ca2+, reduced intracellular ATP and by elevatedconcentrations of long-chain acylcamitines and fatty acids [1, 2]. Afterthe acute phase of ischemia, remodelling occurs where the architectureof the infarcted area and its border zone is altered. This remodellinghas been associated with areas of connexin disarray and the developmentof potential reentrant circuits, which are believed to act asarrhythmogenic substrates [3]. In vivo and in vitro studies have shownthat a group of antiarrhythmic peptides can delay and inhibit the onsetof arrhythmia, and subsequent studies have shown that these peptidesincrease the electrical coupling between cells [2].

A. Methods

1. Cell culture: In short the hearts from 20 neonate rats (age 1-2 days)are excised aseptically. The ventricles are isolated and cut into 4-6pieces, which are digested in several steps in a Hanks buffered salinewith trypsin and DNAse. The cells are then centrifuged and resuspendedin MEM with 5% FCS. To eliminate non-muscle cells the suspension ispreplated in petri dishes for 30 minutes at 37° C. The cells insuspension are then seeded onto collagen coated coverslips. Seedingdensity will be adjusted to give confluent cultures.

2. Electrophysiology: The cover slips with confluent cardiomyocytes aremounted in an open chamber on the stage of an inverted microscope andsuperfused with Dulbeccos phosphate buffered saline (PBS) by gravitydriven flow at 1 ml/min, 37° C. The solution (PBS) contain (in mM): Na+152, K+ 4.2, Cl− 141.5, PO43-9.5, Ca2+ 0.9, Mg2+ 0.5, pH 7.4. Patchclamp pipettes are pulled from 1.5 mm glass capillaries (GC150F-15,Harvard Apparatus) on a Sutter Flaming-Brown P-87 microelectrode pullerand fire polished to a resistance of 4-6 MW. Pipettes are filled with anintracellular like solution containing in mM: K+ 150, Na+ 15, Cl− 5,Gluconate-150.2, EGTA 5, HEPES 5, Ca2+ 2 mM, Mg2+ 1.6, pH 7.2.

The patch clamp set-up consists of a synchronised discontinuousamplifier (SEC-05LX, NPI electronics) and data is digitised using anINT-10 interface (NPI electronics) and a PC1200 data acquisition board(National Instruments). Both current and voltage signals are low passfiltered at 1 kHz using the internal filters of the amplifiers anddigitised at 10 kHz.

A cell is approached with an electrode using a PatchMan 5173micromanipulator (Eppendorf). When contact with the cell is obtained(seen as a sudden increase in input resistance), suction is applieduntil the Giga seal configuration is established. Then a brief pulse ofsuction is applied to break the membrane under the pipette. Theamplifier is then put in the zero-current clamp mode to monitor themembrane potential.

Some distance (

1 cm) away a bipolar platinum electrode is used to pace the culture. Thedelay between the stimulus artifact and the appearance of an actionpotential in the patched cell is then a measure of the conductionvelocity.

The cells were perfused with control solution with 0.1 g/l BSA until astable baseline in the stimulus-activation interval (SAI) wasestablished. Then the perfusate was changed to solution without glucosefor 15 minutes and subsequently challenged with compound 1 (10⁻⁸ M) for20 minutes. In parallel, control experiments were performed which wereconducted on a similar timescale without adding compound 1. Date areexpressed as percent change relative to baseline prior to removal ofglucose from the media.

B. Results

As illustrated in FIG. 2, metabolic stress induced by removal of glucoseproduced a slight increase in the stimulus-activation intervalsuggesting that removal of glucose delayed the conduction velocity. Incontrast, superfusion with a 10 nM concentration of compound 1 decreasedthe stimulus-activation interval, i.e. compound 1 increased conductionvelocity in cultured primary cardiomyocytes. These data indicate thatcompound 1 increases cell-to-cell coupling in cardiomyocytes.

REFERENCES

-   [1] Carmeliet, E. Cardiac ionic currents and acute ischemia: from    channels to arrhythmias. Physiol Rev., 1999, 79: 917-1017.-   [2] Dhein, S. Cardiac gap junctions. Physiology, regulation,    pathophysiology and pharmacology. Karger, Basel, 1998.-   [3] Peters, N. S. and Wit, A. L. Myocardial architecture and    ventricular arrhythmogenesis. Circulation, 1998, 97: 1746-1754.

EXAMPLE 3 Immunoprecipitation and Analysis of Phosphorylated Connexins

Compounds used in this example were made using standard solid phase Fmocchemistry. See the prior examples. Identification was performed by massspectrometry, and the purity, determined by RP-HPLC. In situ binding andanimal studies have shown that the following peptide (compound 1) bindsto the receptor in the nano-molar range. Initial studies with compound 1in 1 nM, 10 nM, 50 nM and 100 nM will determine whether studies with thefollowing peptides compound 3 (H-GAG-4-Hyp-PY-NH2) and compound 4(H-GNY-NH2) are conducted. These studies will also determine whetherstudies involving specific kinase inhibitors are conducted.

H9c2 cells were seeded in 24-multi well dishes in a density of 7,900cells/cm² (˜15,000 cells/well) and grown for 3 days in MEM supplementedwith 10% foetal calf serum (FCS) and 1000 units penicillin/1000 μgstreptomycin (pen/strep) in an atmosphere of 5% CO₂ and 100% humidity at37° C.

The labelled cells were incubated in Triton X-100 lysis buffer (˜5*10⁷cells/ml) for 1 hr at 4° C. The lysate is centrifuged 30 min at 20,000*gfor 30 min. The supernatant was precleared by adding 10 ul Sepharose per200 μl supernatant. The precleared supernatant were then added 0.5-1 μgphospho-tyrosine or phospho-serine antibodies (Sigma) and incubated for1.5 hr at 4° C. in an orbital shaker. The antibody-complex was thencaptured by incubation with 50 μl of a 1:1 slurry of protein G Sepharosefor 1.5 hr. Finally the complex were extensively washed with 0.1% (w/v)Triton X-100, 50 mM Tris, pH 7.4, 300 mM NaCl and 5 mM EDTA.

The complexes were loaded onto a 12% SDS gel and blotted onto a PVDFmembrane. The membrane is blocked with 0.1% Tween 20, 50 mM Tris, pH7.4, 300 mM NaCl, 5% dry milk (TBST) for minimum 2 h. The membrane wassubsequently incubated with connexin 43 specific antibodies (Zymed) for1.5 h at RT and developed with ECL+ (Amersham).

FIGS. 3A-B show immunoblots in which the anti-phosphotyrosine antibodywas used. Cx43 fragment phosphorylation is depicted as a function ofcompound 1 (3A-B) and compound 2 (3B) concentration.

FIGS. 4A-B show immunoblots in which the anti-phosphoserine antibody(4A) or the anti-phosphotyrosine (4B) antibody was used.

EXAMPLE 4 Effect of Compound 1 Peptide on Hemichannel Activity inConfluent Cardiac Myocytes

1. Cell culture: Briefly, the hearts from 20 neonate rats (age 1-2 days)were excised aseptically. The ventricles were isolated and cut into 4-6pieces, which are digested in several steps in a Hanks buffered salinewith trypsin and DNAse. The cells were then centrifuged and resuspendedin MEM with 5% FCS. To eliminate non-muscle cells the suspension waspreplated in petri dishes for 30 minutes at 37° C. The cells insuspension were then seeded onto collagen coated coverslips.

2. Dye-uptake: The cover slips with cardiomyocytes were incubated for 30minutes with control solution (PBS) containing in mM: Na⁺ 152, K⁺ 4.2,Cl⁻ 141.5, PO4³⁻ 9.5, Ca²⁺ 0.9, Mg²⁺ 0.5, glucose 6, pH 7.4. or anischmia mimicking (injury) solution (as control only K⁺ 8.2, no glucose,10 mM deoxy-glucose, pH 6.5), both solutions containing 200 μM calcein.Then the cells were washed for 10 minutes with control solution.

The cover slips were mounted in an open chamber on the stage of aninverted microscope. Ten random fields were chosen and from each fieldthree cells were chosen under conventional light microscopy. Then thecells were excited by 480 nm light and the fluorescence emmissionmeasured at 510 nm, where the emission intensity is a measure ofdye-uptake.

The effect of the compound 1 on dye-uptake was investigated under twoconditions:

Experiment 1: In this series of experiments the cells were be incubatedwith control solution with calcein for 30 minutes and washed for 10minutes without dye. In paired experiments the same experiment wasperformed where the peptide is added during the 30 minutes incubation.

Experiment 2: As experiment 1 only performed with injury solutioninstead of control solution.

3. Results Experiments using uptake of calcein in culturedcardiomyocytes from neonate rats show that the dye uptake is 38% higherthan in control cells, when the cells are exposed to metabolic stress(in the form of low pH, increased extracellular K⁺ and deoxyglucose 5-10mM). This effect could be significantly reduced to 5% when the cellswere co-incubated with compound 1 (10 nM, P<0.017 in paired t-testversus cells exposed to deoxyglucose alone, n=6). These results showthat the increased membrane permeability through connexin hemi-channelsseen after metabolic stress can be inhibited by compound 1. It is likelythat this effect will be beneficial for cellular function and survival.Furthermore, phosphorylation experiments on cultured H9c2 cardiomyocyteshave shown that compound 1 mediates tyrosine phosphorylation of connexin43. Moreover, Cardiomyocytes exposed for 4 h with 0, 1, 10, 50 and 100nM compound 1 an increase connexin 43 tyrosine phosphorylation. Tyrosinephosphorylation has been reported to mediate a disruption of gapjunction intercellular communication and could easily explain thedecrease in membrane permeability seen in the calcein experiments. Insome experiments, the compound 1 was found to decreaseSer-phosphorylation.

FIG. 5 shows effects of 10 nM compound 1 on ischemia-induced uptake ofthe calcein in the cultured cardiomyocytes.

EXAMPLE 5 ELISA Assay of Site-Specific Connexin Phosphorylation

An ELISA sandwich assay has been developed which enables measurements ofsite-specific phosphorylation of connexins in tissue samples as well asin cell cultures in a multi-well format (24-96 wells). Wells are coatedwith antibody (capture antibody) against the connexin type in question.Cell or tissue extracts are then reacted with capture antibody-coatedplates at 4° C. o/n. The captured connexins that are phosphorylated aredetected with an antibody directed against specific phosphorylationsites and conjugated with either biotin, FITC, TRITC or peroxidase.

The amounts of antibody bound to the specific phosphorylation sites mayalternatively be measured with a radioactive labelled or an enzymeconjugated antibody against the species IgG of the detection antibody.

The principle has been demonstrated using mouse antiCx43 as captureantibody, rabbit anti-Tyr-P (tyrosine phosphorylation site) as detectionantibody and anti-rabbit IgG either labelled with ¹²⁵I, or HRP (horseradish peroxidase) to measure the amounts of bound anti-Tyr-P. Theprinciple may also be demonstrated using rabbit antiCx43 (5 μg/ml; cat.no. 710700, Zymed Lab. Inc., California, USA) as capture antibody, amonoclonal mouse anti-phosphotyrosine (50 μg/ml; cat. no. P-3300, clonept66, Sigma, Mo., USA) as detection antibody and anti-mouse wholeantibody either labelled with ¹²⁵I, (sheep anti-mouse Ig cat. no. IM131, Amersham Biosciences, Wales, UK) or peroxidase (donkey anti-mouseIg; cat. no. 715-035-151) to measure the amounts of boundanti-phosphotyrosine.

FIG. 6A shows in schematic form a preferred ELISA detection format.FIGS. 6B and 6C show results of this example.

The present example and discussion provides evidence that metabolicstress opens connexin hemi-channels in cardiac cells, and that thisopening can be circumvented by incubation with compound 1. Moreover,Examples 2-5 show that compound 1 closes hemichannels during metabolicstress. In agreement with the inhibitory effect of compound 1 on calceinuptake by hemichannels, i.e. a closing of hemichannels, the Examplesalso show that compound 1 assists in modulating the tyrosinephosphorylation state of connexin 43. Compound 1 is known to open gapjunctions, cf. WO01/62775 and it is known that the inhibition ofdephosphorylation of connexins prevents closing of gap junctionchannels. Thus, it is believed that compounds provided herein which canbe shown to have beneficial gap junction opening or modulating effect ofcompound 1, i.e. all antiarrhythmic peptides disclosed in WO01/62775 andknown peptides such as AAP, AAP10, HP5 and the like are useful in thepresent invention as modulators of hemichannel opening or closing. It isalso believed that compound 1 maintains or increases the tyrosinephosphorylation of Cx43, thereby modulating hemichannel function.

EXAMPLE 6 Dye Uptake in Cardiomyocytes

1. Methods Ventricular myocytes were isolated from neonate rats bytrypsin digestion and plated onto collagen-coated cover slips. Afterfour days the myocytes formed confluent and synchronously beating sheetsof cells. To measure dye uptake, cells were incubated in buffercontaining 200 μM calcein for 30 minutes at room temperature. Thencoverslips were thoroughly washed with control buffer and mounted in anopen bath chamber and superfused with control buffer (RT). Cells wereexcited with 480 nm light by means of a Xenon lamp and a monochromator.Images of the fluorescence emitted at 510 nm were collected by a cooledCCD camera (Sensicam). Excitation control and image acquisition wascontrolled using Imaging workbench (Axon).

Under light microscopy three regions were placed over cardiomyocytes inan image. Then average fluorescence intensity was measured, and theprocedure was repeated until 30 regions in 10 images had been measured.The average of these measurements were used as value for the givenexperimental condition, that is n=1. In each series one controlincubation was performed and all experimental values are given asrelative to this value.

Solutions: Control buffer (in mM): NaCl 136, KCl 4, MgCl₂ 0.8, CaCl₂ 1.8HEPES 5, MES 5, Glucose 6, pH 7.3 Stress buffer (in mM): NaCl 136, KCl8, MgCl₂ 0.8, CaCl₂ 1.8 HEPES 5, MES 5, Deoxy-glucose 10, pH 6.2

2. Results Incubation of cells with calcein under control condition gavesome fluorescence staining, which was mostly of a localized particulatenature (see FIG. 7A-C, panel B). On top of the cultured myocytes, curledup cells that immediately stained with trypan blue, were often found.These cells stained heavily with calcein and care was always taken notto place the regions of measurements over these cells. When cells wereexposed to metabolic inhibition the pattern of staining changed, anddiffuse staining throughout the cytosol was observed (FIG. 7C). Theaverage intensity during stress was 36.2±4.18% higher compared tocontrol (n=5, P<0.001 in paired t-test).

This confirms findings in cardiac and other cell types, that metabolicinhibition activates the uptake of fluorescent dyes, probably viaconnexin hemi-channels. We hypothesized that one of the mechanisms bywhich the AAP family of peptides could exert its positive effects oncardiac tissue, could be to interrupt activation of hemi-channels,thereby improving cellular homeostasis. To investigate this, cells wereexposed to stress in the presence of ZP123 in concentrations between 0.1pM and 10 nM. The results are presented in FIG. 8, where each data pointrepresent 5 experiments. As can be seen ZP123 dose-dependently reducedthe uptake of calcein. Using a sigmoidal function ED₅₀ was estimated to3.3±5.3 pM with a Hill coefficient of 2.5±1.0. Average intensity duringmaximal inhibition by compound 1 was 6.9±1.7% above control.

EXAMPLE 7 Volume Measurements in Cardiomyocytes

1. Methods: Ventricular myocytes were isolated from neonate rats bytrypsin digestion and plated onto collagen-coated cover slips. Afterfour days the myocytes formed confluent and synchronously beating sheetsof cells. To measure cell volume cells were loaded with calcein-AM (5 μMin control buffer) for 15 minutes at 37° C. and the cover slips mountedin an open bath chamber. The following experiments were performed atroom temperature. Using a Leica laser confocal microscope, an opticalcross-section was performed every 20 seconds throughout the duration ofthe experiments.

The optical cross-sections were analyzed using Metamorph (UniversalImaging). The area of the cell was determined as the area of pixels withfluorescence intensities higher than a threshold value chosen tominimize background induced fluctuations. Data are presented as relativevolumes by dividing each value by the average area under controlconditions (the last measurement before metabolic stress).

Solutions: Control buffer (in mM): NaCl 136, KCl 4, MgCl₂ 0.8, CaCl₂ 1.8HEPES 5, MES 5, Glucose 6, pH 7.3 Stress buffer (in mM): NaCl 136, KCl8, MgCl₂ 0.8, CaCl₂ 1.8 HEPES 5, MES 5, Deoxy-glucose 10, pH 6.2

2. Results Under control conditions cells on average tended to slightlydecrease their volume with time (FIG. 9B shows average data from 5experiments). When cells were challenged with metabolic inhibition, thefall in volume was reversed to a net-increase (filled squares in FIG.9A). On average the relative volume was 1.06±0.02 (last five minutes ofstress, n=5), whereas the volume of control cells in this period was0.94±0.04.

Dye uptake measurements in myocytes have shown that compound 1 inhibitsopen connexin hemi-channels. It is believed that the hemi-channels mightcontribute to the observed swelling. To test this compound 1 (0.1 nM)was included in the stress medium and volume monitored. Data from sixexperiments are shown in FIG. 9A (open squares) and as can be seen thestress induced swelling was prevented. Average relative volume duringstress in the presence of compound 1 was 0.93±0.05.

When comparing data from control, stress and stress plus compound 1using a one-way ANOVA, significant differences were detected (see FIG.10). Post.hoc. testing (LSD) showed that stress was significantlydifferent from control (P<0.05) and stress plus compound 1 (P<0.05),whereas control was not different from stress plus compound 1 (P>0.76).

EXAMPLE 8 Reduction of Infarct Size after Myocardial Infarction in Rats

1. Methods:

Male Lewis rats (300-350 g; M&B, Ll. Skendsved, Denmark) wereanaesthetized with a neurolept anaesthesic combination (Hypnorm®(fentanyl citrate 0.315 mg/ml and fluanisone 10 mg/ml)+midazolam (5mg/ml)). Commercial solution of midazolam was diluted 1:2 in distilledwater. Three parts of the diluted midazolam is mixed with one parthypnorm®. Anesthesia was induced by s.c. administration of 0.2 ml ofthis solution per 100 gram rat. When surgical anesthesia wasestablished, an endotracheal cannula is inserted and the animal isartificially ventilated using a Harvard rodent ventilator adjusted tomaintain arterial pH at 7.3-7.5 during surgery.

Compound 1 was delivered by an osmotic minipump (Alzet model 2ML4) thatwas inserted into the intraperitoneal (i.p.) cavity immediately prior toinduction of the myocardial infarction. The pump was filled with vehicle(isotonic saline) or Compound 1 and primed at 37° C. for 24 hours priorto the operation. In order to ensure that the therapeutic plasmaconcentration was obtained already at the time of infarction, a loadingdose of Compound 1 was given s.c. prior to ligation of the LAD. With thes.c. loading dose, a steady-state plasma level was reached already after3 min.

After administration of the s.c. loading dose of Compound 1 and afteri.p. insertion of the osmotic minipump, a left thoracotomy wasperformed, and the left anterior descending artery was ligated using a6-0 silk suture. The thorax is closed in layers, and a negative pressurewas induced in the pleural cavity to unfold the lungs. Sham-operatedanimals were subjected to the same procedure without ligation of theLAD. Postoperatively, the animal were allowed to recover in a heatedcabinet at 30° C. with 50% oxygen until the next morning. To preventdehydration during the recovery period, the rat was given 5 ml 5%glucose s.c. immediately after completion of surgery and another 5 ml 5%glucose at 4 p.m. To relieve postoperative pain, the rat was treatedwith buprenorphine (20 μg/100 g s.c. b.i.d.) and meloxicam (0.1 mg/100 gs.c. once daily) for three days after the myocardial infarction. In thisstudy the infusion dose given to rats with myocardial infarction (MI)was adjusted to produce plasma concentrations at 0 [vehicle=isotonicsaline], 1.7±0.4 nM [dose 1], 5.7±0.4 [dose 2], or 93.7±12.3 nM [dose3]. Plasma concentrations of Compound 1 were measured by massspectrometry following solid phase extraction. Doses used are shownbelow in Table I: TABLE I Plasma S.c. loading concentration dose of I.p.infusion after 3 weeks Compound 1 dose of Compound 1 Group nM (nmol/kg)(pmol/kg/min) Sham-MI 0 0 0 MI 0 0 0 MI 1.7 ± 0.4 0.25 11 MI 5.7 ± 0.42.5 110 MI 93.7 ± 12.3 25 1100

Three weeks after ligation of LAD, the rats were anesthetized with ani.p, injection of pentobarbital (50 mg/kg). The animals were placed on aheating blanket in order to maintain body temperature at 37° C.Tracheotomy was performed and the rat was ventilated with oxygen using aHarvard rodent ventilator. The ventilator was adjusted to maintainarterial pH at 7.35-7.45. PE50 catheters were placed in the femoral veinand artery for i.v. administration and arterial pressure measurements,respectively. After insertion of the femoral i.v. catheter, the animalwas paralysed by i.v. administration of pancuronium, 1 mg/kg.Anaesthesia and respiratory paralysis is maintained by continuous i.v.infusion of pentobarbital (2.5 mg/kg/h) and pancuronium (1 mg/kg/h)delivered in isotonic saline (50 μl/min). A tygon catheter was insertedinto the left ventricle via the right carotid artery for determinationof left ventricular end-diastolic pressure. After recording of leftventricular end-diastolic pressure (LVEDP), The hearts are removed fromthe bodies by cutting the veins and arteries a few mm from the hearts.The hearts were carefully rinsed for blood with isotonic saline, weighedand placed in 4% neutral buffered formalin.

After fixation the large vessels were cut close to the heart. Thereafterthe hearts were cut into parallel slices with a thickness of 1.5 mmusing an aggregate consisting of parallel razor blades with a distanceof 1.5 mm. The slices are perpendicular to the long axis of the heart.

The slices were placed in two histological capsules so that the firstslice is placed in one of the capsules by random number and the secondin the other, the third in the first capsule and so on. After embedding,the slices were cut into 4 μm thick sections that are stained byHematoxylin-eosin and by Masson-trichrome.

Using a microscope with a stage that can be moved in predeterminedsteps, both Masson-trichrome stained slides were examined and the numberof points hitting fibrous tissue (blue stained in Masson-trichrome) andnormal tissue are counted. The microscope was connected to a videocamera and the image is displayed on a screen. The morphometry systemused was Cast2 from Olympus, Denmark. Infarct size was calculated as %fibrous tissue in the heart.

2. Results:

As illustrated in FIG. 11, the heart weight-body weight ratio wasreduced by the two highest doses of Compound 1 suggesting that thecompound reduces the hypertrophic consequences (i.e., remodelling) ofmyocardial infarction.

As shown in FIG. 12, treatment with Compound 1 for three weeks reducedinfarct size by 20-50% relative to infarct size in rats subjected to amyocardial infarction, but treated with vehicle. This indicates thatCompound 1 has cytoprotective actions during myocardial ischemia. Inline with what has been described herein, these data indicate thatCompound 1 reduces the size of the myocardial infarct by a mechanismthat may involve prevention of cell swelling. Thus, prevention of cellswelling may prevent compression of surrounding healthy tissue, andtherefore prevent compression of the microcirculation in surroundinghealthy tissue. Therefore, in this application we claim that thisprinciple may prevent the consequence of any ischemic lesion in anyorgan, preferably but not limited to organs with a confined fibrouscapsule (e.g., heart, kidney) or organs surrounded by bone (e.g., brain,spinal cord, bone marrow).

As shown in FIG. 13, rats subjected to myocardial infarction but treatedwith either dose of Compound 1 for three weeks, had better an improvedcardiac function with less congestion in the left ventricle asdemonstrated by a reduced left ventricular end-diastolic pressure. Thisdata indicate that Compound 1, prevents the impairment of cardiacfunction following a myocardial infarction.

All references disclosed herein are incorporated by reference. Theinvention has been described with reference to preferred embodimentsthereof. However, it will be appreciated that those skilled in the art,upon consideration of this disclosure, may make modifications andimprovements within the spirit and scope of the invention.

1. A method of modulating a hemichannel in a cell, tissue or organexposed to stress, the method comprising contacting the stressed cell,tissue or organ with a therapeutically effective amount of Compound 1,wherein the contact is sufficient to close the hemichannel in thestressed cell, tissue or organ, and wherein Compound 1 is defined asAc-D-Tyr-D-Pro-D-4Hyp-Gly-D-Ala-Gly-NH₂.
 2. The method of claim 1,wherein the method further comprises phosphorylating a tyrosine residueof connexin 43 (Cx 43).
 3. A method of preventing or treating tissue ororgan stress in a mammal, the method comprising administering atherapeutically effective amount Compound 1, wherein the contact issufficient to prevent or treat the stress in tissue or organ, andwherein Compound 1 is defined asAc-D-Tyr-D-Pro-D-4Hyp-Gly-D-Ala-Gly-NH₂.
 4. A method of increasing gapjunction intracellular communication (GJIC) in a cell, tissue or organ,the method comprising administering a therapeutically effective amountof Compound 1, wherein the contact is sufficient to increase the GJIC inthe cell, tissue or organ, and wherein Compound 1 is defined asAc-D-Tyr-D-Pro-D-4Hyp-Gly-D-Ala-Gly-NH₂.
 5. A method of treatment ofburns comprising administering to a patient in need of such treatment atherapeutically effective amount of Compound 1, wherein Compound 1 isdefined as Ac-D-Tyr-D-Pro-D-4Hyp-Gly-D-Ala-Gly-NH₂.
 6. A method oftreatment of thromboses comprising administering to a patient in need ofsuch treatment a therapeutically effective amount of Compound 1, whereinCompound 1 is defined as Ac-D-Tyr-D-Pro-D-4Hyp-Gly-D-Ala-Gly-NH₂.
 7. Amethod of treatment of respiratory and metabolic acidosis comprisingadministering to a patient in need of such treatment a therapeuticallyeffective amount of Compound 1, wherein Compound 1 is defined asAc-D-Tyr-D-Pro-D-4Hyp-Gly-D-Ala-Gly-NH₂.
 8. A method of treatment offocal arrhythmia comprising administering to a patient in need of suchtreatment a therapeutically effective amount of Compound 1, whereinCompound 1 is defined as Ac-D-Tyr-D-Pro-D-4Hyp-Gly-D-Ala-Gly-NH₂.
 9. Amethod of treating and preventing cell and tissue damage resulting fromelevated levels of blood glucose comprising administering to a patientin need of such treatment a therapeutically effective amount of Compound1, wherein Compound 1 is defined asAc-D-Tyr-D-Pro-D-4Hyp-Gly-D-Ala-Gly-NH₂.
 10. A method of treatment ofchronic atrial fibrillation comprising administering to a patient inneed of such treatment a therapeutically effective amount of Compound 1,wherein Compound 1 is defined asAc-D-Tyr-D-Pro-D-4Hyp-Gly-D-Ala-Gly-NH₂.
 11. A method of cytoprotectingtissue or an organ of a mammal in need of such treatment, the methodcomprising administering a therapeutically effective amount of Compound1, wherein Compound 1 is defined asAc-D-Tyr-D-Pro-D-4Hyp-Gly-D-Ala-Gly-NH₂.
 12. A method of preventing ortreating reperfusion injury in a mammal, the method comprisingadministering a therapeutically effective amount of Compound 1, whereinCompound 1 is defined as Ac-D-Tyr-D-Pro-D-4Hyp-Gly-D-Ala-Gly-NH₂. 13.The method of claim 3 or claim 14, wherein the method further comprisesphosphorylating a tyrosine residue of connexin 43 (Cx 43) and closingthe hemichannel.
 14. A method of preventing or treating tissue or organstress in a mammal, the method comprising administering atherapeutically effective amount of at least one compound selected fromthe group consisting of compounds represented as Formula I or II asdescribed above, wherein the contact is sufficient to prevent or treatthe stress in tissue or organ, and wherein Formula I is defined asfollows:

wherein, R1 represents H or acetyl (Ac) R2 represents a sidechain of oneof the amino acids Gly, Tyr, D-Tyr, Phe and D-Phe, R3 represents anyamino acid sidechain, R4 represents a sidechain of one of the aminoacids Gly, Tyr, D-Tyr, Phe and D-Phe, R5 represents OH or NH2, and a, S,T, P and Q are integers and independently=0 or 1; and salts thereof, andwherein Formula II is defined as follows:R1-X1-X2-X3-R2 wherein, X1 is 0, Ala, Gly, β-Ala, Tyr, D-Tyr, Asp, X2 is0, Ala-Gly-T4c-Pro, Ala-Sar-Hyp-Pro, Ala-Asn, D-Asn-D-Ala, D-Asn, Gly,Ala, D-Ala, β-Ala, Asn or, X3 is Tyr, D-Tyr, Gly or Phe, and R1 is H orAc, R2 is OH or NH2, with the proviso that X1 and X2 are not both 0; andsalts thereof.
 15. A method of increasing gap junction intracellularcommunication (GJIC) in a cell, tissue or organ, the method comprisingadministering a therapeutically effective amount of at least onecompound selected from the group consisting of compounds represented asFormula I or II, wherein the contact is sufficient to increase the GJICin the cell, tissue or organ, and wherein Formula I is defined asfollows:

wherein, R1 represents H or acetyl (Ac) R2 represents a sidechain of oneof the amino acids Gly, Tyr, D-Tyr, Phe and D-Phe, R3 represents anyamino acid sidechain, R4 represents a sidechain of one of the aminoacids Gly, Tyr, D-Tyr, Phe and D-Phe, R5 represents OH or NH2, and a, S,T, P and Q are integers and independently=0 or 1; and salts thereof, andwherein Formula II is defined as follows:R1-X1-X2-X3-R2 wherein, X1 is 0, Ala, Gly, β-Ala, Tyr, D-Tyr, Asp, X2 is0, Ala-Gly-T4c-Pro, Ala-Sar-Hyp-Pro, Ala-Asn, D-Asn-D-Ala, D-Asn, Gly,Ala, D-Ala, β-Ala, Asn or, X3 is Tyr, D-Tyr, Gly or Phe, and R1 is H orAc, R2 is OH or NH2, with the proviso that X1 and X2 are not both 0; andsalts thereof.
 16. A method of treatment of burns comprisingadministering to a patient in need of such treatment a therapeuticallyeffective amount of a compound according to Formula I or II that blocksconnexin hemichannel opening, wherein Formula I is defined as follows:

wherein, R1 represents H or acetyl (Ac) R2 represents a sidechain of oneof the amino acids Gly, Tyr, D-Tyr, Phe and D-Phe, R3 represents anyamino acid sidechain, R4 represents a sidechain of one of the aminoacids Gly, Tyr, D-Tyr, Phe and D-Phe, R5 represents OH or NH2, and a, S,T, P and Q are integers and independently=0 or 1; and salts thereof, andwherein Formula II is defined as follows:R1-X1-X2-X3-R2 wherein, X1 is 0, Ala, Gly, β-Ala, Tyr, D-Tyr, Asp, X2 is0, Ala-Gly-T4c-Pro, Ala-Sar-Hyp-Pro, Ala-Asn, D-Asn-D-Ala, D-Asn, Gly,Ala, D-Ala, β-Ala, Asn or, X3 is Tyr, D-Tyr, Gly or Phe, and R1 is H orAc, R2 is OH or NH2, with the proviso that X1 and X2 are not both 0; andsalts thereof.
 17. A method of treatment of thromboses comprisingadministering to a patient in need of such treatment a therapeuticallyeffective amount of a compound according to Formula I or II that blocksconnexin hemichannel opening, and wherein Formula I is defined asfollows:

wherein, R1 represents H or acetyl (Ac) R2 represents a sidechain of oneof the amino acids Gly, Tyr, D-Tyr, Phe and D-Phe, R3 represents anyamino acid sidechain, R4 represents a sidechain of one of the aminoacids Gly, Tyr, D-Tyr, Phe and D-Phe, R5 represents OH or NH2, and a, S,T, P and Q are integers and independently=0 or 1; and salts thereof, andwherein Formula II is defined as follows:R1-X1-X2-X3-R2 wherein, X1 is 0, Ala, Gly, β-Ala, Tyr, D-Tyr, Asp, X2 is0, Ala-Gly-T4c-Pro, Ala-Sar-Hyp-Pro, Ala-Asn, D-Asn-D-Ala, D-Asn, Gly,Ala, D-Ala, β-Ala, Asn or, X3 is Tyr, D-Tyr, Gly or Phe, and R1 is H orAc, R2 is OH or NH2, with the proviso that X1 and X2 are not both 0; andsalts thereof.
 18. A method of treatment of respiratory and metabolicacidosis comprising administering to a patient in need of such treatmenta therapeutically effective amount of a compound according to Formula Ior II that blocks connexin hemichannel opening, wherein Formula I isdefined as follows:

wherein, R1 represents H or acetyl (Ac) R2 represents a sidechain of oneof the amino acids Gly, Tyr, D-Tyr, Phe and D-Phe, R3 represents anyamino acid sidechain, R4 represents a sidechain of one of the aminoacids Gly, Tyr, D-Tyr, Phe and D-Phe, R5 represents OH or NH2, and a, S,T, P and Q are integers and independently=0 or 1; and salts thereof, andwherein Formula II is defined as follows:R1-X1-X2-X3-R2 wherein, X1 is 0, Ala, Gly, β-Ala, Tyr, D-Tyr, Asp, X2 is0, Ala-Gly-T4c-Pro, Ala-Sar-Hyp-Pro, Ala-Asn, D-Asn-D-Ala, D-Asn, Gly,Ala, D-Ala, β-Ala, Asn or, X3 is Tyr, D-Tyr, Gly or Phe, and R1 is H orAc, R2 is OH or NH2, with the proviso that X1 and X2 are not both 0; andsalts thereof.
 19. A method of treatment of focal arrhythmia comprisingadministering to a patient in need of such treatment a therapeuticallyeffective amount of a compound according to Formula I or II that blocksconnexin hemichannel opening, wherein Formula I is defined as follows:

wherein, R1 represents H or acetyl (Ac) R2 represents a sidechain of oneof the amino acids Gly, Tyr, D-Tyr, Phe and D-Phe, R3 represents anyamino acid sidechain, R4 represents a sidechain of one of the aminoacids Gly, Tyr, D-Tyr, Phe and D-Phe, R5 represents OH or NH2, and a, S,T, P and Q are integers and independently=0 or 1; and salts thereof, andwherein Formula II is defined as follows:R1-X1-X2-X3-R2 wherein, X1 is 0, Ala, Gly, β-Ala, Tyr, D-Tyr, Asp, X2 is0, Ala-Gly-T4c-Pro, Ala-Sar-Hyp-Pro, Ala-Asn, D-Asn-D-Ala, D-Asn, Gly,Ala, D-Ala, β-Ala, Asn or, X3 is Tyr, D-Tyr, Gly or Phe, and R1 is H orAc, R2 is OH or NH2, with the proviso that X1 and X2 are not both 0; andsalts thereof.
 20. A method of treating and preventing cell and tissuedamage resulting from elevated levels of blood glucose comprisingadministering to a patient in need of such treatment a therapeuticallyeffective amount of a compound according to Formula I or II that blocksconnexin hemichannel opening, wherein Formula I is defined as follows:

wherein, R1 represents H or acetyl (Ac) R2 represents a sidechain of oneof the amino acids Gly, Tyr, D-Tyr, Phe and D-Phe, R3 represents anyamino acid sidechain, R4 represents a sidechain of one of the aminoacids Gly, Tyr, D-Tyr, Phe and D-Phe, R5 represents OH or NH2, and a, S,T, P and Q are integers and independently=0 or 1; and salts thereof, andwherein Formula II is defined as follows:R1-X1-X2-X3-R2 wherein, X1 is 0, Ala, Gly, β-Ala, Tyr, D-Tyr, Asp, X2 is0, Ala-Gly-T4c-Pro, Ala-Sar-Hyp-Pro, Ala-Asn, D-Asn-D-Ala, D-Asn, Gly,Ala, D-Ala, β-Ala, Asn or, X3 is Tyr, D-Tyr, Gly or Phe, and R1 is H orAc, R2 is OH or NH2, with the proviso that X1 and X2 are not both 0; andsalts thereof.
 21. A method of treatment of chronic atrial fibrillationcomprising administering to a patient in need of such treatment atherapeutically effective amount of a compound according to Formula I orII that blocks connexin hemichannel opening, and wherein Formula I isdefined as follows:

wherein, R1 represents H or acetyl (Ac) R2 represents a sidechain of oneof the amino acids Gly, Tyr, D-Tyr, Phe and D-Phe, R3 represents anyamino acid sidechain, R4 represents a sidechain of one of the aminoacids Gly, Tyr, D-Tyr, Phe and D-Phe, R5 represents OH or NH2, and a, S,T, P and Q are integers and independently=0 or 1; and salts thereof, andwherein Formula II is defined as follows:R1-X1-X2-X3-R2 wherein, X1 is 0, Ala, Gly, β-Ala, Tyr, D-Tyr, Asp, X2 is0, Ala-Gly-T4c-Pro, Ala-Sar-Hyp-Pro, Ala-Asn, D-Asn-D-Ala, D-Asn, Gly,Ala, D-Ala, β-Ala, Asn or, X3 is Tyr, D-Tyr, Gly or Phe, and R1 is H orAc, R2 is OH or NH2, with the proviso that X1 and X2 are not both 0; andsalts thereof.
 22. A method of treatment of epilepsia comprisingadministering to a patient in need of such treatment a therapeuticallyeffective amount of a compound according to Formula I or II thatpromotes connexin hemichannel opening, and wherein Formula I is definedas follows:

wherein, R1 represents H or acetyl (Ac) R2 represents a sidechain of oneof the amino acids Gly, Tyr, D-Tyr, Phe and D-Phe, R3 represents anyamino acid sidechain, R4 represents a sidechain of one of the aminoacids Gly, Tyr, D-Tyr, Phe and D-Phe, R5 represents OH or NH2, and a, S,T, P and Q are integers and independently=0 or 1; and salts thereof, andwherein Formula II is defined as follows:R1-X1-X2-X3-R2 wherein, X1 is 0, Ala, Gly, β-Ala, Tyr, D-Tyr, Asp, X2 is0, Ala-Gly-T4c-Pro, Ala-Sar-Hyp-Pro, Ala-Asn, D-Asn-D-Ala, D-Asn, Gly,Ala, D-Ala, β-Ala, Asn or, X3 is Tyr, D-Tyr, Gly or Phe, and R1 is H orAc, R2 is OH or NH2, with the proviso that X1 and X2 are not both 0; andsalts thereof.
 23. A method of cytoprotecting tissue or an organ of amammal in need of such treatment, the method comprising administering atherapeutically effective amount of at least one compound selected fromthe group consisting of the compounds represented by Formula I or II,and wherein Formula I is defined as follows:

wherein, R1 represents H or acetyl (Ac) R2 represents a sidechain of oneof the amino acids Gly, Tyr, D-Tyr, Phe and D-Phe, R3 represents anyamino acid sidechain, R4 represents a sidechain of one of the aminoacids Gly, Tyr, D-Tyr, Phe and D-Phe, R5 represents OH or NH2, and a, S,T, P and Q are integers and independently=0 or 1; and salts thereof, andwherein Formula II is defined as follows:R1-X1-X2-X3-R2 wherein, X1 is 0, Ala, Gly, β-Ala, Tyr, D-Tyr, Asp, X2 is0, Ala-Gly-T4c-Pro, Ala-Sar-Hyp-Pro, Ala-Asn, D-Asn-D-Ala, D-Asn, Gly,Ala, D-Ala, β-Ala, Asn or, X3 is Tyr, D-Tyr, Gly or Phe, and R1 is H orAc, R2 is OH or NH2, with the proviso that X1 and X2 are not both 0; andsalts thereof.
 24. The method of claim 11 or claim 23, wherein themethod further comprises exposing the tissue or organ of the mammal toischemic conditions.
 25. The method of claim 24, wherein the organ to becytoprotected is associated with a fibrous capsule or bone.
 26. Themethod of claim 25, wherein the organ is heart, kidney, brain, spinalcord or bone marrow.
 27. The method of claim 26, wherein the heart hasbeen subjected to an infarction and the ischemia is associated withmyocardial cell swelling.
 28. The method of claim 27, wherein thecompound is Ac-Gly-Asn-Tyr-NH₂ (Compound 2).
 29. A method of preventingor treating reperfusion injury in a mammal, the method comprisingadministering a therapeutically effective amount of at least onecompound selected from the group consisting of the compounds representedby Formula I or II, and wherein Formula I is defined as follows:

wherein, R1 represents H or acetyl (Ac) R2 represents a sidechain of oneof the amino acids Gly, Tyr, D-Tyr, Phe and D-Phe, R3 represents anyamino acid sidechain, R4 represents a sidechain of one of the aminoacids Gly, Tyr, D-Tyr, Phe and D-Phe, R5 represents OH or NH2, and a, S,T, P and Q are integers and independently=0 or 1; and salts thereof, andwherein Formula II is defined as follows:R1-X1-X2-X3-R2 wherein, X1 is 0, Ala, Gly, β-Ala, Tyr, D-Tyr, Asp, X2 is0, Ala-Gly-T4c-Pro, Ala-Sar-Hyp-Pro, Ala-Asn, D-Asn-D-Ala, D-Asn, Gly,Ala, D-Ala, β-Ala, Asn or, X3 is Tyr, D-Tyr, Gly or Phe, and R1 is H orAc, R2 is OH or NH2, with the proviso that X1 and X2 are not both 0; andsalts thereof.
 30. The method of claim 12 or claim 29, wherein themethod further comprises exposing the heart of the mammal to infarctconditions and establishing coronary perfusion.
 31. The method of claim30, wherein the method further comprises administering a thrombolyticagent or providing coronary angioplasty to facilitate coronary perfusioninto the infarcted heart.
 32. The method of claim 31, wherein thecompound is Ac-Gly-Asn-Tyr-NH₂ (Compound 2).